Systems, methods and compositions for optimizing tissue and cell enriched grafts

ABSTRACT

Disclosed herein are methods and systems for the concentration of cells from a cell suspension into unprocessed tissue, such as adipose tissue. Also disclosed herein are systems for optimizing hydration of tissue and cell enriched grafts.

The present application claims priority to U.S. Provisional ApplicationSer. No. 61/174,860, filed on May 1, 2009, by Peterson et al., andentitled “SYSTEMS, METHODS AND COMPOSITIONS FOR OPTIMIZING TISSUE ANDCELL GRAFTS,” the entire disclosure of which is herein expresslyincorporated by reference in its entirety.

FIELD OF THE INVENTION

Embodiments disclosed herein generally relate to compositions thatcomprise grafts, implants, or transplantable preparations comprisingadipose tissue with and without a population of adipose-derivedregenerative cells (e.g., a concentrated population of adipose-derivedregenerative cells that comprise stem cells) and methods and systems forpreparing, optimizing and administering the same.

BACKGROUND OF THE INVENTION

The transfer of adipose tissue to various regions of the body is arelatively common cosmetic, therapeutic and structural procedureinvolving the harvest of adipose tissue from one location andre-implantation of the harvested and, oftentimes processed tissue, inanother location (see Coleman 1995; and Coleman 2001). While beinglargely used for repair of small cosmetic defects such as facial folds,wrinkles, pock marks and divots; the transfer of adipose tissue hasrecently been used for cosmetic and/or therapeutic breast augmentationand reconstruction (Bircoll and Novack 1987; and Dixon 1988), andaugmentation of the buttocks (Cardenas-Camarena, Lacouture et al. 1999;de Pedroza 2000; and Peren, Gomez et al. 2000).

In the past, adipose tissue grafts and methods of adipose tissuetransfer have been plagued with difficulties and side effects includingnecrosis, absorption of the implant by the body, infection (Castello,Barros et al. 1999; Valdatta, Thione et al. 2001), calcifications andscarring (Huch, Kunzi et al. 1998), inconsistent engraftment, (Eremiaand Newman 2000), lack of durability, and other problems arising fromlack of neovascularization and necrosis of the transplanted tissue. Oneof the biggest challenges in adipose tissue transfer is absorption ofthe implant by the body and volume retention of adipose tissue graftsfollowing transfer. When adipose tissue is harvested or washed, thespace between individual pieces of harvested adipose tissue is filled byliquid (e.g., water, blood, tumescent solution, oil). When thistissue/fluid mixture is implanted into a recipient the liquid portion israpidly absorbed by the body resulting in loss of volume. The process bywhich the amount of fluid is removed from the tissue/fluid mixture isfrequently referred to as “drying the adipose tissue” or “dehydratingthe adipose tissue”. The content of red and white blood cells and thelike within an adipose tissue graft can also significantly affect thevolume of graft retained after graft transplantation, due to inductionor exacerbation of an inflammatory response. Another aspect of tissueretention relates t the amount of lipid within the adipose tissue graft.It understood that the presence of free lipid (meaning lipids releasedfrom dead or damaged adipocytes; also referred to as oil) in adiposetissue grafts can result in induction or exacerbation of an inflammatoryresponse with substantial phagocytic activity and consequent loss ofgraft volume.

It is also known that mixing unprocessed adipose tissue with aconcentrated population of adipose-derived regenerative cells overcomesmany of the problems associated with adipose tissue grafts and adiposetissue transfer, as described above. Specifically, supplementingunprocessed adipose tissue with concentrated populations ofadipose-derived cells comprising adipose-derived stem cells increasesthe weight, vascularization, and retention of fat grafts. (See U.S. Pat.No. 7,390,484 and co-pending U.S. Patent Application Publication No.2005/0025755, herein expressly incorporated by reference in theirentireties). Adipose tissue fragments supplemented, or mixed, with aconcentrated population of cells including adipose-derived stem cellsexhibit improved neoangiogeneis and perfusion in grafts when compared tounsupplemented grafts of adipose tissue alone in animal models. Further,adipose tissue grafts supplemented with adipose-derived regenerativecells that comprise adipose derived stem cells show increased graftretention and weight over time, when compared to unsupplemented grafts.(See U.S. Patent Application Publication No. 2005/0025755). Further, theprocessing of adipose tissue in a closed, sterile fluid pathway greatlyreduces the chance of infection. The improvement in autologous transferof adipose tissue seen in the animal models described above has alsobeen replicated in human clinical studies. Nevertheless, the isolationand purification of concentrated populations of adipose-derivedregenerative cells comprising adipose-derived stem cells (ADSCs),usually involves a series of washing, digestion, filtration and/orcentrifugation steps, which can reduce the yield of viable cells,require mechanical equipment and specialized clinicians, and/or cancompromise the quality, appearance, longevity, hydration or efficacy ofthe graft.

The need for additional approaches to prepare and optimize adiposetissue grafts and implants and to isolate and/or concentrateadipose-derived regenerative cells is manifest.

SUMMARY OF THE INVENTION

Embodiments described herein relate to devices to process adipose tissuegrafts, as well as approaches for preparation of adipose tissue graftsand adipose tissue grafts supplemented with adipose-derived regenerativecells.

Several embodiments provided herein concern devices for preparing tissuefor an adipose tissue graft. In some embodiments, the device can includea flexible, collapsible bag having a first chamber and a second chamberwhich are defined by a filter having pores. The device can also includea separator located within the second chamber, and one or more inletports and an outlet port connected to the flexible, collapsible bag. Theinlet port can be configured to allow the aseptic introduction ofadipose tissue into the first chamber; and the outlet port can beconfigured to aseptically remove liquid and cells from the secondchamber.

In some embodiments, the separator can be a free floating porousstructure within the second chamber. In some embodiments, the separatorcan be porous structure that defines a third chamber within the secondchamber. In some embodiments, the separator can include a lipid-wickingmaterial, such as a polyester mesh screen or the like. In someembodiments the separator can be a porous structure, having pores thatare larger than the pores of the filter of the system. For example, insome embodiments, the pores of the separator have a pore size that canbe greater than or equal to about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, or 40 times the pore size of the pores of thefilter. In some embodiments, the pore size of the separator can bebetween about 300 and 2000 μm. In some embodiments, the pore size of thefilter can be greater than or equal to about 30 μm, e.g., between about30 μm and about 200 μm. In some embodiments, the pore size of the filteris 35 μm.

In some embodiments, the inlet port can be configured to releasablyconnect with an adapter. In some embodiments, the adapter can beconfigured to releasably connect with the tip of a syringe barrel, suchas a 60 or a 250 ml syringe, a Toomey syringe, or the like. In someembodiments, the inlet port can be configured to allow material to enterinto the port, but not to exit from the port. For example, in someembodiments, the inlet port includes or is configured to be coupled to adeformable plastic valve and/or a tissue access port assembly. In someembodiments, the inlet port can be configured to be attached to acannula, while maintaining a sterile fluid/tissue pathway.

In some embodiments, the device can include a second device, wherein thesecond device is an adipose-derived regenerative cell isolation device.In some embodiments, the adipose-derived regenerative cell isolationdevice can be attached to the first device for preparing tissue for anadipose tissue graft while maintaining a closed pathway. In someembodiments, the adipose-derived regenerative cell isolation device canbe a device as described herein above. In some embodiments, the seconddevice can be connected to the device for preparing tissue for anadipose tissue graft by a conduit that can be configured to transferisolated adipose-derived regenerative cells from the second device tothe first chamber of the device for preparing tissue for an adiposetissue graft. In some embodiments, the conduit can include a Yconnection.

Some embodiments provided herein relate to a method of making an adiposetissue graft. The method can include the steps of obtaining a firstportion of unprocessed adipose tissue; rinsing the first portion ofunprocessed adipose tissue with a physiologic solution; and dehydratingthe rinsed adipose tissue to an amount of hydration that is less thanthe amount of hydration present in the first portion of unprocessedadipose tissue prior to dehydration. For example, in some embodiments,the rinsed adipose tissue is dehydrated to a liquid content that is lessthan about ½, ⅓, or ¼ times (preferably about ⅓ times) that of saidfirst portion of unprocessed adipose tissue prior to dehydration.

In some embodiments, the physiologic solution can be Lactated Ringer'ssolution, Ringer's acetate, saline, phosphate buffered saline,PLASMALYTE™ solution, crystalloid solutions and IV fluids, colloidsolutions and IV fluids, five percent dextrose in water (D5W),Hartmann's Solution or the like.

In some embodiments, the method can additionally include the steps ofisolating a population of adipose-derived regenerative cells from asecond portion of adipose tissue and contacting the dehydrated adiposetissue with the isolated population of adipose-derived regenerativecells under conditions that allow the isolated population ofadipose-derived regenerative cells to permeate through the dehydratedadipose tissue. In some embodiments, the isolated population ofadipose-derived regenerative cells is not subjected to centrifugationprior to contacting the dehydrated adipose tissue. In some embodiments,the isolated population of adipose-derived regenerative cells can beprepared in a device disclosed herein above, by contacting adiposetissue present in the first chamber of the device with a means ofreleasing cells from the connective tissue matrix, for example, andenzyme solution comprising collagenase under conditions that liberatesaid cells. In some embodiments, such conditions that liberate saidcells include heat, cooling, mechanical digestion, ultrasound or laserassisted liberation or other methods known in the art and described U.S.Pat. No. 7,390,484, which is expressly incorporated herein in itsentirety. In some embodiments, the contacting step can be performed in asecond device (e.g. a device having the same structure as the firstdevice) attached to the first device.

Some embodiments relate to a method of producing an adipose tissue graftincluding the steps of obtaining a first portion of unprocessed adiposetissue introducing the first portion of unprocessed adipose tissue intothe first chamber of a device described above; adding a physiologic washsolution to the first chamber with the unprocessed adipose tissue torinse the unprocessed adipose tissue; and removing fluid (e.g., water,physiologic wash solution, blood, free lipid, or the like, or anycombination thereof), from the second chamber of the device, therebydrying the adipose tissue and reducing the free lipid content.

In some embodiments, the method can additionally include the steps ofisolating a population of adipose-derived regenerative cells from asecond portion of adipose tissue and contacting the dehydrated adiposetissue with the isolated population of adipose-derived regenerativecells under conditions that allow the isolated population ofadipose-derived regenerative cells to permeate through the dehydratedadipose tissue. In some embodiments, the isolated population ofadipose-derived regenerative cells is not subjected to centrifugationprior to contacting the dehydrated adipose tissue. In some embodiments,the isolated population of adipose-derived regenerative cells of isprepared in a device disclosed herein by contacting adipose tissuepresent in the first chamber of the device with a means of releasingcells from the connective tissue matrix, for example, and enzymesolution comprising collagenase under conditions that liberate thecells. In some embodiments, such conditions that liberate said cellsinclude heat, cooling, mechanical digestion, ultrasound or laserassisted liberation or other methods known in the art and described U.S.Pat. No. 7,390,484, which is expressly incorporated herein in itsentirety. In some embodiments, the contacting step can be performed in asecond device (e.g. a device having the same structure as the firstdevice) attached to the first device.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram illustrating exemplary graft supplementationmethods disclosed herein.

FIG. 2 shows a block diagram of a flexible collection container.

FIG. 3 illustrates an exemplary system used to optimize an adiposetissue graft. The graft may be supplemented with adipose derivedregenerative cells.

FIG. 4 illustrates ports and adaptors in system 300.

FIG. 5 illustrates the outer membrane 310 of system 300.

FIG. 6 illustrates the filter 320 of system 300.

FIG. 7 illustrates the separation screen 330 of system 300.

FIG. 8 illustrates exemplary seals 311 of system 300.

FIG. 9 is a perspective view of a system 300 with ports and adaptors600.

FIG. 10 is a cutaway view of a tissue port assembly 600 used with theports of system 300.

FIG. 11 is a cutaway view of a tissue port assembly 600 used with theports of system 300.

FIG. 12 is a cutaway view of a cap 610 used with a tissue port assembly600.

FIG. 13 is an illustration of an exemplary system 800 used to optimizean adipose tissue graft.

FIGS. 14A and 14B are perspective views of an exemplary tissueenrichment device. FIG. 14A shows a canister with a first chamber forprocessing adipose tissue, and a second chamber, for supplementingadipose tissue with lipodigestate, for grafting. FIG. 14B depicts anexemplary housing/platform for the canister shown in FIG. 14A.

FIG. 15 is a bar graph showing the percent water content (v/v) ofunprocessed adipose tissue (control), adipose tissue prepared by agravity preparation method (Gravity), adipose tissue prepared by acentrifugation method (Centrifugation) and dried adipose tissue preparedaccording to the methods and systems described herein (PureGraft).

FIG. 16 is a bar graph showing the percent lipid content (v/v) ofunprocessed adipose tissue (control), adipose tissue prepared by agravity preparation method (Gravity), adipose tissue prepared by acentrifugation method (Centrifugation) and dried adipose tissue preparedaccording to the methods and systems described herein (PureGraft).

FIG. 17 is a bar graph showing the content of red blood cells (RBC) pergram of tissue normalized to the red blood cell content present per gramof tissue within grafts prepared according to the methods and systemsdescribed herein (PureGraft). Data are shown for unprocessed adiposetissue (control), adipose tissue prepared by a gravity preparationmethod (Gravity), and adipose tissue prepared by a centrifugation method(Centrifugation), and adipose tissue prepared according to the methodsand systems described herein (PureGraft).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Embodiments disclosed herein relate to methods and systems for theproduction of adipose tissue grafts (e.g., “fat grafts”) or adiposetissue implants, either alone or supplemented, enhanced, or fortifiedwith adipose-derived regenerative cells (e.g., a cell population thatcomprises adipose-derived stem cells, endothelial cells and/orprogenitor cells). The embodiments disclosed herein are based, in part,on the discovery of a device or system that can be used for the rapidpreparation and optimization of adipose tissue grafts, implants, and forthe preparation of grafts and implants enriched with a population ofadipose-derived regenerative cells (e.g., a cell population comprisingadipose derived stem cells) by, for example, gravity flow and, ifdesired, in the absence of centrifugation, high pressure, or vacuumfiltration. As discussed herein below, the adipose tissue graftsprepared using the devices disclosed herein have reduced levels offluid, blood cells, and free lipid or oil content compared to graftsprepared using conventional techniques. Notably, the adipose tissuegrafts prepared using the devices disclosed herein need not be subjectedto strong mechanical forces, which may lead to decreased cell viabilityand reduced retention of the adipose tissue graft. Using the devicesdisclosed herein, one can obtain a more predictable and stableadipose-tissue implant.

The embodiments disclosed herein are also based, in part, on Applicants'discovery that intact adipose tissue fragments or “unprocessed adiposetissue matrix” can be used to filter, bind and thereby effectivelyconcentrate in situ the adipose-derived regenerative cells that areprovided vis a vis a solution or suspension of digested or partially orfully disaggregated adipose tissue. In some embodiments, “dried adiposetissue” or “dehydrated adipose tissue” can be used to filter, bind andthereby effectively concentrate in situ adipose-derived regenerativecells provided in the form of lipo-digestate. In some embodiments, the“unprocessed adipose tissue matrix” can be dried or dehydrated afteradding the lipo-digestate. Accordingly, it has been realized that insome embodiments, concentration of the cellular component ofdisaggregated adipose tissue prior to augmentation, supplementation, orfortification of an adipose tissue graft or fat graft is no longerrequired.

Reference will now be made in detail to the presently preferredembodiments of the invention, examples of which are illustrated in theaccompanying drawings. Wherever possible, the same or similar referencenumbers are used in the drawings and the description to refer to thesame or like parts. It should be noted that the drawings are insimplified form and are not to precise scale. In reference to thedisclosure herein, for purposes of convenience and clarity only,directional terms, such as, top, bottom, left, right, up, down, over,above, below, beneath, rear, and front, are used with respect to theaccompanying drawings. Such directional terms should not be construed tolimit the scope of the invention in any manner.

Although the disclosure herein refers to certain illustratedembodiments, it is to be understood that these embodiments are presentedby way of example and not by way of limitation. The intent of thefollowing detailed description, although discussing exemplaryembodiments, is to be construed to cover all modifications,alternatives, and equivalents of the embodiments as may fall within thespirit and scope as defined by the appended claims. Aspects of thepresent invention may be practiced in conjunction with various cell ortissue separation techniques that are conventionally used in the art,and only so much of the commonly practiced process steps are includedherein as are necessary to provide an understanding of the presentinvention.

Dried or Dehydrated Adipose Tissue

Some embodiments provided herein relate to methods of producing dried ordehydrated adipose tissue grafts that can be used directly in autologoustransplantation procedures, e.g., autologous transplantation, or thatcan be fortified with cells (e.g., adipose-derived regenerative cells,adipose-derived stem cells, or the like), additives or the like prior totransplantation.

The term “adipose tissue,” in some contexts, may refer to fat includingthe connective tissue that stores fat. Adipose tissue contains multipleregenerative cell types, including adipose-derived stem cells(“ADSCs”),endothelial progenitor and precursor cells, pericytes,macrophages, fibroblasts, lymphatic cells including lymphaticendothelial cells, etc., bound up by the connective tissue matrix. Insome embodiments, a unit of adipose tissue is removed from a subject togenerate an adipose tissue graft.

A “unit of adipose tissue” refers to a discrete or measurable amount ofadipose tissue, which can be measured by determining the weight and/orvolume of the unit. A unit of adipose tissue may refer to the entireamount of adipose tissue removed from a patient, or an amount that isless than the entire amount of adipose tissue removed from a patient.Thus, a unit of adipose tissue may be combined with another unit ofadipose tissue to form a unit of adipose tissue that has a weight orvolume that is the sum of the individual units.

In some embodiments, one or more units of adipose tissue is/are removedfrom a subject. The adipose tissue used in the embodiments describedherein can be obtained by any method known to a person of ordinary skillin the art. For example, adipose tissue can be removed from a patient bysuction-assisted lipoplasty, ultrasound-assisted lipoplasty, excisionallipectomy, laser lipoplasty, water jet lipoplasty, or the like. Inaddition, the procedures may include a combination of such procedures,such as a combination of excisional lipectomy and suction-assistedlipoplasty. Preferably, the adipose tissue is collected in a manner thatpreserves the viability of the tissue and its cellular component andminimizes the likelihood of contamination of the collected material withpotentially infectious organisms, such as bacteria and/or viruses. Thus,in preferred embodiments, the tissue extraction is performed in asterile or aseptic manner to minimize contamination, e.g., in a closedsterile fluid/tissue pathway. In some embodiments, suction assistedlipoplasty is used to remove the adipose tissue from a patient, therebyproviding a minimally invasive method of collecting tissue with reducedpotential for cell or tissue damage that may be associated with othertechniques, such as ultrasound assisted lipoplasty.

For suction-assisted lipoplastic procedures, adipose tissue is collectedby insertion of a cannula into or near an adipose tissue depot presentin the subject followed by aspiration of the adipose into a suctiondevice. In one embodiment, a small cannula can be coupled to a syringe,and the adipose tissue can be aspirated using manual force. Using asyringe or other similar device can be used to harvest relativelymoderate amounts of adipose tissue (e.g., from 0.1 ml to several hundredmilliliters of adipose tissue). Procedures employing these relativelysmall devices have the advantage that the procedures can be performedwith only local anesthesia, as opposed to general anesthesia. Largervolumes of adipose tissue above this range (e.g., greater than severalhundred milliliters) may require general anesthesia at the discretion ofthe donor and the person performing the collection procedure. Whenlarger volumes of adipose tissue are desired to be removed, relativelylarger cannulas and automated suction devices can be employed in theprocedure.

Excisional lipectomy procedures include, and are not limited to,procedures in which adipose tissue-containing tissue (e.g., skin) isremoved by excision such as surgical dissection under direct or indirectvisualization of the tissue being excised. In certain embodiments thismay occur as an incidental part of the procedure; that is, where theprimary purpose of the surgery is the removal of tissue (e.g., skin inbariatric or cosmetic surgery) and in which adipose tissue is removedalong with the tissue of primary interest.

The amount of adipose tissue collected for use in the methods disclosedherein is dependent on a number of variables including, but not limitedto, the body mass index of the donor, the availability of accessibleadipose tissue harvest sites, concomitant and pre-existing medicationsand conditions (such as anticoagulant therapy), and the purpose forwhich the tissue is being collected. Engraftment of adipose tissuetransplants has been shown to be cell dose-dependent with thresholdeffects. Thus, it is likely that the general principle that “more isbetter” will be applied within the limits set by other variables andthat where feasible the harvest will collect as much tissue as possible.

In some embodiments, e.g. in embodiments wherein the dried or dehydratedadipose tissue is used to make a fortified or supplemented adiposetissue graft, a unit of adipose tissue is divided into portions. Thefirst portion of the adipose tissue is not digested, and is either notprocessed at all, or rinsed or washed to obtain dried or dehydratedadipose tissue as described herein below. The first portion ofunprocessed, dried, or dehydrated adipose tissue can serve as the graftfoundation that is supplemented with adipose-derived regenerative cells(e.g., a cell population that comprises adipose-derived stem cells,and/or endothelial cells and/or progenitor cells) present in thelipo-digestate or concentrated population of adipose-derived cells fromthe second portion. One portion can be processed as described below torelease or liberate the adipose-derived regenerative cells (e.g., a cellpopulation that comprises adipose-derived stem cells, and/or endothelialcells and/or progenitor cells) from the connective tissue matrix toobtain a lipo-digestate, or concentrated population of adipose-derivedcells comprising regenerative cells or stem cells. In some embodiments,two different units of adipose tissue are collected, e.g., from the sameor different regions of the subject, or from different subjects. Oneunit can be processed as described below to obtain lipo-digestate or aconcentrated population of adipose-derived cells comprising regenerativecells or stem cells, and the other unit can serve as the foundation forthe fat graft that is supplemented with adipose-derived regenerativecells (e.g., lipo-digestate and/or a cell population that comprisesadipose-derived stem cells, and/or endothelial cells and/or progenitorcells). In one embodiment one or both of the units of adipose tissue maybe cryopreserved such that delivery of the graft to the patient may beseparated in time from harvest of the tissue. In one such embodiment oneor both units of tissue may be cryopreserved within the device or systemof the present invention wherein the chambers of the device arefabricated from materials that retain mechanical and structuralintegrity during the processes of cryopreservation, cryostorage,thawing, and subsequent use as described herein. In another suchembodiment the lipodigestate or concentrated population ofadipose-derived cells comprising regenerative cells including adiposederived stem cells may be cryopreserved prior to use in fortification ofthe graft or implant as described herein.

The term “dried,” as used in reference to “dried adipose tissue,” refersto a unit of adipose tissue having a lower content of liquid, e.g.,water or other liquid (e.g., tumescent fluid), present in the “driedadipose tissue” as compared to unprocessed adipose tissue from the samesite and same subject (e.g., an equivalent unit (w/w) of adipose tissuetaken from the same site and same subject as the adipose tissue that wasdried). The term “dehydrated,” as used in reference to “dehydratedadipose tissue,” refers to a unit of adipose tissue having a lowercontent of liquid, e.g., water or other liquid (e.g., tumescent fluid),present in the “dried adipose tissue” as compared to unprocessed adiposetissue from the same site and same subject (e.g., an equivalent unit(w/w) of adipose tissue taken from the same site and same subject as theadipose tissue that was dried).

The term “equivalent unit” as used herein can refer to an equivalentvolume or weight of adipose tissue obtained from a subject. For example,an equivalent unit can mean an equivalent volume (or weight) of adiposetissue obtained from a subject. In some embodiments, an equivalent unitcan mean an equivalent volume (or weight) obtained from the same site(e.g., buttocks, abdomen, thigh, back, or the like) from the same or adifferent subject.

“Unprocessed adipose tissue” refers to adipose tissue that has not beenpartially or fully disaggregated, i.e., by subjecting the tissue tomechanical and/or enzymatic disaggregation. As such, unprocessed tissuecontains intact tissue fragments, that include connective tissue boundto adipose-derived regenerative cells. As used herein, “adipose-derivedregenerative cell” refers to any cells obtained from adipose tissuewhich cause or contribute to complete or partial regeneration,restoration, or substitution of structure or function of an organ,tissue, or physiologic unit or system to thereby provide a therapeutic,structural or cosmetic benefit. Examples of regenerative cells include:adipose-derived stem cells (“ADSCs”), endothelial cells, endothelialprecursor cells, endothelial progenitor cells, macrophages, fibroblasts,pericytes, smooth muscle cells, preadipocytes, differentiated orde-differentiated adipocytes, keratinocytes, unipotent and multipotentprogenitor and precursor cells (and their progeny), and lymphocytes.

In some embodiments, dried or dehydrated adipose tissue has about 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 20%, 25%, 30%, 35% 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% (or any % inbetween this range) of the liquid content (as measured by volume and/orweight), of an equivalent unit of unprocessed adipose tissue. Forexample, dried adipose tissue can have greater than or equal to about1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8times, 9 times, or 10 times (or any number in between this range) lessliquid content than an equivalent unit of unprocessed adipose tissue.Similarly, the term “dehydrated,” as used in reference to “dehydratedadipose tissue,” refers to a lower content of water present in the“dehydrated adipose tissue,” as compared to unprocessed adipose tissue,or an equivalent unit of unprocessed adipose tissue. In someembodiments, dehydrated adipose tissue can have greater than or equal toabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 20%, 25%, 30%,35% 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90% or 95% (or any % inbetween this range) of the water contentof an equivalent unit ofunprocessed adipose tissue, or an equivalent unit of adipose tissueprepared by a centrifugation or another conventional approach. Forexample, dried adipose tissue can have greater than or equal to about1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8times, 9 times, or 10 times (or any number in between this range) lesswater content than an equivalent unit of unprocessed adipose tissue.

In some embodiments, “dried” or “dehydrated” adipose tissue describedherein can contain a lower content or percentage of lipid and/or red orwhite blood cells compared to an equivalent unit of unprocessed adiposetissue. In some embodiments, dried or dehydrated adipose tissue can haveat less than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%,20%, 25%, 30%, 35% 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, 95% or99% (or any % in between this range) (or any number in between thisrange) of the white blood cells in an equivalent unit of unprocessedadipose tissue and/or an equivalent unit of adipose tissue processedusing a centrifugation method, e.g., wherein the excised tissue is spunin a fixed angle centrifuge or another conventional preparationtechnique. For example, in some embodiments, dried or dehydrated adiposetissue can contain less than about 75%, less than 80%, less than 85%,less than 90%, less than 95%, or less, or any % in between this range,of the number of white blood cells in an equivalent unit of adiposetissue.

In some embodiments, the dried or dehydrated adipose tissue providedherein can have at less than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 12%, 15%, 20%, 25%, 30%, 35% 40%, 45%, 50%, 55%, 60%, 65%, 70%,80%, 90% or 95% (or any % in between this range) (or any number inbetween this range) of the red blood cells in an equivalent unit ofunprocessed adipose tissue, or adipose tissue prepared using acentrifugation method, e.g., wherein the excised tissue is spun in afixed angle centrifuge, or by another conventional preparative approach.For example, in some embodiments, the adipose tissue grafts produced inthe systems disclosed herein contain less than about 75%, less than 80%,less than 85%, less than 90%, less than 95%, or less, or any % inbetween this range, of the number of red blood cells in an equivalentunit of adipose tissue.

In some embodiments, the dried or dehydrated adipose tissue graftsdisclosed herein have less than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 12%, 15%, 20%, 25%, 30%, 35% 40%, 45%, 50%, 55%, 60%, 65%, 70%,80%, 90% or 95% (or any % in between this range) (or any number inbetween this range) of lipid in an equivalent unit of unprocessedadipose tissue, or adipose tissue prepared by a conventionalcentrifugation protocol, e.g., wherein the excised adipose tissue isspun in a fixed angle centrifuge. For example, in some embodiments, thedried or dehydrated adipose tissue grafts disclosed herein contain lessthan about 75%, less than 80%, less than 85%, less than 90%, less than95%, or less, or any % in between, of the percentage of free lipidcontent present in an equivalent unit of unprocessed adipose tissue.

In some embodiments, dried or dehydrated adipose tissue is obtained byproviding unprocessed adipose tissue in a device that includes a filterand a separator, as described in further detail below. The filter candivide the device into two internal chambers, thereby defining a firstchamber and a second chamber or subsystem. The adipose tissue isintroduced into the first chamber or subsystem of the device, andpreferably does not enter into the second chamber of the device. Thefilter has a plurality of pores, that allow for the free flow of liquidsuch as, water, tumescent fluid, wash solution, (e.g., Lactated Ringers,saline, PLASMALTYE™ and the like), free lipid, oil, blood cells, lysedcells from the adipose tissue and blood components, into the secondchamber, but the pore size is such that it retains non-disaggregatedadipose tissue and tissue fragments in the first chamber. The secondchamber can include a separator made from material that is lipid-wickingand/or fluid-wicking (e.g., a meshwork design that draws fluid from thefirst chamber), as described in further detail below. In preferredembodiments, the separator is made from a porous material, wherein thepores of the separator are larger than the pores of the filter.

The unprocessed adipose tissue within the first chamber is rinsed orwashed with a physiologic wash solution. In preferred embodiments, thephysiologic wash solution is aseptically introduced into the device. Insome embodiments, the wash solution is introduced into the firstchamber. In some embodiments, the wash solution is introduced into thesecond chamber, and passes through the filter into the second chamber,thereby coming in contact with the adipose tissue therein. In someembodiments, the wash solution is introduced into both the first and thesecond chambers.

In some embodiments, the adipose tissue and the wash solution areagitated (e.g., by inverting, squeezing, or rocking the device gently),in order to facilitate the rinsing and separation of free lipid, red andwhite blood cells, and tumescent fluid from the adipose tissue in thefirst chamber. In other embodiments, the adipose tissue within the firstchamber is contacted with the wash solution, which is allowed to drainor be drawn from the first chamber of the device without agitation,e.g., by gravitational or wicking forces.

In some embodiments, the volume of wash solution used to rinse theadipose tissue can be greater than the volume of the adipose tissue. Byway of example only, in some embodiments, greater than or equal to about1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, 20ml, 25 ml, 30 ml, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml,400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 650 ml, 700 ml, 750 ml, 800 ml,850 ml, 900 ml, 950 ml, 1000 ml 1100 ml, 1500 ml, 2000 ml or any amountin between these volumes, of wash solution can be used to rinse theunprocessed adipose tissue.

During the washing or rinsing step, liquid, e.g. wash solution, freelipid, oil, blood cells, lysed cells from the adipose tissue and bloodcomponents, and the like pass through the pores of the filter betweenthe first and second chambers of the device. As such, the second chamberbecomes filled with liquid. The separator within the second chamber canfunction to draw and retain fluid from the first chamber into the secondchamber, e.g., acting as a wick. The movement of water, tumescent fluid,blood and free lipid from the first chamber, which houses the adiposetissue, dries and dehydrates the adipose tissue. The fluid is removedfrom the second chamber through a port. In some embodiments, fluid isremoved from the second chamber using a pump or a vacuum. In someembodiments, fluid is allowed to drain from the port leaving the secondchamber.

Exemplary devices for making dried or dehydrated adipose tissue, as wellas, supplemented or fortified adipose tissue grafts are discussed infurther detail below, with reference to FIGS. 2-13.

In some embodiments, the steps of adding wash solution and removingcontents from the second chamber is repeated 1 time, 2 times, 3 times, 4times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more.

The dried or dehydrated adipose tissue can be removed (preferablyaseptically) from the first chamber, and administered to a subjectdirectly. In some embodiments, the dried or dehydrated adipose tissue isprocessed further (e.g., by the addition of an additive, as described infurther detail below), prior to administration to a subject.

Supplemented Adipose Tissue Grafts

Described herein are methods and systems for producing supplemented,enhanced, or fortified adipose tissue grafts, e.g., wherein the graft isunprocessed adipose tissue or dried or dehydrated adipose tissue. Forexample, in some methods described herein, the fortified or supplementedadipose tissue grafts are supplemented with additional adipose-derivedregenerative cells or adipose-derived stem cells, e.g., withlipo-digestate or a concentrated adipose-derived cell populationcomprising regenerative cells or stem cells. Preferably, the additionaladipose-derived regenerative cells or adipose-derived stem cells areobtained from the same subject. In some embodiments, the additionaladipose-derived regenerative cells or adipose derived stem cells can befrom a different subject.

By some of the methods described herein, for example, digestedlipoaspirate (“lipo-digestate”) is applied directly onto unprocessedadipose tissue, dried adipose tissue, dehydrated adipose tissue, orunprocessed adipose tissue matrix, and the unprocessed adipose tissue,dried adipose tissue, dehydrated adipose tissue, or unprocessed adiposetissue matrix is used as a filter or sieve to retain components presentin the lipo-digestate (e.g., adipose-derived regenerative cells, such asa cell population that comprises adipose-derived stem cells and/orendothelial cells and/or progenitor cells). By this process, one canrapidly prepare an adipose tissue graft, implant, or fat graft that hasbeen enriched, supplemented or fortified with said adipose-derivedregenerative cells (e.g., a cell population that comprisesadipose-derived stem cells, and/or endothelial cells and/or progenitorcells), without additional purification or isolation steps, which may becumbersome, time consuming, and may have an impact on cell viability. Bysome of the methods described herein, for example, concentratedpopulations of adipose-derived cells comprising regenerative cells orstem cells is applied directly onto unprocessed adipose tissue, driedadipose tissue, dehydrated adipose tissue, or unprocessed adipose tissuematrix, and the unprocessed adipose tissue, dried adipose tissue,dehydrated adipose tissue.

In contrast to existing approaches to isolate, purify, and concentrateadipose-derived regenerative cells, some methods disclosed herein usethe intact matrix of the unprocessed adipose tissue, dried adiposetissue, or dehydrated adipose tissue to gently filter and concentratethe adipose-derived regenerative cells in situ, that is, on the matrixitself and by doing so avoid the cell damage brought about bycentrifugation, membrane, gel, or gradient, filtration and othermechanical manipulations of lipo-digestate. Additionally, the approachdescribed herein promotes an even or substantially complete distributionof the exogenous adipose-derived regenerative cells (e.g., a cellpopulation that comprises adipose-derived stem cells, and/or endothelialcells and/or progenitor cells) throughout the adipose tissue graft.

As used herein, “regenerative cell composition” or “lipo-digestate”refers to the composition of cells typically present in a volume ofliquid after a tissue, e.g., adipose tissue, is washed and at leastpartially disaggregated. For example, in some embodiments, aregenerative cell composition or lipo-digestate can comprise a cellsolution that comprises a population of adipose-derived cells thatcomprises adipose-derived regenerative cells, e.g., stem cells. In someembodiments, regenerative cell compositions can include multipledifferent types of regenerative cells, including ADSCs, endothelialcells, endothelial precursor cells, endothelial progenitor cells,macrophages, fibroblasts, pericytes, smooth muscle cells, preadipocytes,differentiated or de-differentiated adipocytes, keratinocytes, unipotentand multipotent progenitor and precursor cells (and their progeny), andlymphocytes. In some embodiments, regenerative cell compositions includeonly one, only two, only three, only four or more, types of regenerativecells. Regenerative cell compositions and lipo-digestates can, in someembodiments, also contain one or more contaminants, such as collagen,which may be present in the tissue fragments. In some embodiments, thelipo-digestate, or regenerative cell solution, is substantially free ofintact adipose tissue fragments.

As used herein, “stem cell” refers to a multipotent regenerative cellwith the potential to differentiate into a variety of other cell types,which perform one or more specific functions and have the ability toself-renew. Some of the stem cells disclosed herein may be pluripotent.

As used herein, “progenitor cell” refers to a multipotent regenerativecell with the potential to differentiate into more than one cell type.“Progenitor cell”, as used herein, also refers to a unipotentregenerative cell with the potential to differentiate into only a singlecell type, which performs one or more specific functions and has limitedor no ability to self-renew. In particular, as used herein, “endothelialprogenitor cell” refers to a multipotent or unipotent cell with thepotential to differentiate into vascular endothelial cells.

As used herein, “precursor cell” refers to a unipotent regenerative cellwith the potential to differentiate into one cell type. Precursor cellsand their progeny may retain extensive proliferative capacity, e.g.,lymphocytes and endothelial cells, which can proliferate underappropriate conditions.

As used herein “stem cell number” or “stem cell frequency” refers to thenumber of colonies observed in a clonogenic assay in which adiposederived cells (ADC) are plated at low cell density (<10,000 cells/well)and grown in growth medium supporting MSC growth (for example, DMEM/F12medium supplemented with 10% fetal calf serum, 5% horse serum, andantibiotic/antimycotic agents. Cells can be grown for two weeks afterwhich cultures can be stained with hematoxylin. Colonies of more than 50cells are counted as CFU-F. Stem cell frequency is calculated as thenumber of CFU-F observed per 100 nucleated cells plated (for example; 15colonies counted in a plate initiated with 1,000 nucleated ADC cellsgives a stem cell frequency of 1.5%). Stem cell number is calculated asstem cell frequency multiplied by the total number of nucleated ADCcells obtained. A high percentage (˜100%) of CFU-F grown from ADC cellsexpress the cell surface molecule CD105 which is also expressed bymarrow-derived stem cells (Barry et al., 1999). CD105 is also expressedby adipose tissue-derived stem cells (Zuk et al., 2002). In someembodiments, adipose tissue can be processed according to the methodsdescribed herein to obtain lipo-digestate, and/or a concentratedpopulation of adipose-derived cells, wherein at least 0.2%, 0.3%, 0.4%,0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2%, 3%, 4%, 5%, 6%, 7% 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells arestem cells or other type of regenerative cells of the lipo-digestate orconcentrated adipose-derived cell population.

Preferably, adipose tissue is processed to produce lipo-digestate or aregenerative cell solution in a sterile, closed system, with a closedfluid/tissue pathway, to avoid any contact with the external environmentand eliminate the possibility of contamination from the environment.Devices useful for processing adipose tissue and producinglipo-digestate are known in the art. In preferred embodiments, adiposetissue is processed to produce lipo-digestate while maintaining acompletely closed system using, for example, in some embodiments, adevice as described in U.S. Pat. No. 7,390,484, which is herebyexpressly incorporated by reference in its entirety. In some preferredembodiments, the adipose tissue processing procedure does not includecentrifugation, elutriation, or any other mechanical approaches forconcentrating the cell population comprising adipose-derivedregenerative cells, which causes or has the potential to cause decreasedviability of the regenerative cells in the regenerative cellcomposition/lipo-digestate.

In some embodiments, the process to obtain lipo-digestate includes theremoval or depletion of the tissue of the mature fat-laden adipocytecomponent from the portion or unit of adipose tissue used to produce thelipo-digestate or concentrated adipose-derived cell populations. In someembodiments, the adipose tissue is subjected to a series of washing anddisaggregation steps in which the tissue is first rinsed to reduce thepresence of free lipids (released from ruptured adipocytes) andperipheral blood elements (released from blood vessels severed duringtissue harvest). For example, in some embodiments, the adipose tissue ismixed with isotonic saline, e.g., phosphate buffered saline, or otherphysiologic solution(s) (e.g., PLASMALYTE®, of Baxter Inc., NORMOSO® ofAbbott Labs, or Lactated Ringers solution). The washed tissue can thenbe disaggregated to free intact adipocytes and other cell populationsfrom the connective tissue matrix. In certain embodiments, the entireadipocyte component, or non-regenerative cell component, is separatedfrom the regenerative cell component of the adipose tissue. In otherembodiments, only a portion or portions of the adipocyte component isseparated from the regenerative cells. Intact adipose tissue fragmentscan be separated from the free lipid and cells by several approachesincluding, but not limited to, filtration, decantation, orsedimentation, or the like. Preferably, the digested tissue is notsubjected to centrifugation or elutriation.

In some embodiments, the adipose tissue used to generate thelipo-digestate is fully disaggregated, whereas in other embodiments, itis only partially disaggregated. Intact adipose tissue fragments, e.g.,from unprocessed or washed adipose tissue, can be disaggregated usingany conventional techniques or methods, including mechanical force(mincing or shear forces), enzymatic digestion with single orcombinatorial proteolytic enzymes, such as collagenase, trypsin, lipase,liberase HI, as disclosed in U.S. Pat. No. 5,952,215, and pepsin, or acombination of mechanical and enzymatic methods. Additional methodsusing collagenase that may be used to disaggregate adipose tissue aredisclosed in U.S. Pat. Nos. 5,830,714 and 5,952,215, and by Williams, S.K., S. McKenney, et al. (1995). “Collagenase lot selection andpurification for adipose tissue digestion.” Cell Transplant 4(3): 281-9.In some embodiments, neutral proteases can be used to disaggregatetissue, instead of or in addition to, collagenase, as disclosed inTwentyman, P. R. and J. M. Yuhas (1980). “Use of bacterial neutralprotease for disaggregation of mouse tumours and multicellular tumorspheroids.” Cancer Lett 9(3): 225-8. In some embodiments, adipose tissueis disaggregated with a combination of enzymes, such as a combination ofcollagenase and trypsin, as disclosed in Russell, S. W., W. F. Doe, etal. (1976). “Inflammatory cells in solid murine neoplasms. Tumordisaggregation and identification of constituent inflammatory cells.”Int J Cancer 18(3): 322-30. In some embodiments, adipose tissue can bedisaggregated using a combination of an enzyme, such as trypsin, andmechanical dissociation, as disclosed in Engelholm, S. A., M.Spang-Thomsen, et al. (1985). “Disaggregation of human solid tumours bycombined mechanical and enzymatic methods.” Br J Cancer 51(1): 93-8.

In some embodiments, a portion of the adipose tissue is fullydisaggregated, to separate the adipose-derived regenerative cells (e.g.,adipose-derived stem cells) from the mature adipocytes and connectivetissue. In some embodiments, a portion of the adipose tissue is onlypartially disaggregated. For example, partial disaggregation may beperformed with one or more enzymes, which are removed from the at leasta part of the adipose tissue early, relative to an amount of time thatthe enzyme would otherwise be left thereon to fully disaggregate theportion of the adipose tissue. Such a process may require lessprocessing time.

In some embodiments, a portion or unit of adipose tissue is washed withsterile buffered isotonic saline and incubated with collagenase at acollagenase concentration, temperature, and time sufficient to provideadequate disaggregation. Preferably, enzymes used for disaggregation areapproved for human use by the relevant authority (e.g., the U.S. Foodand Drug Administration), and are free from microorganisms andcontaminants, such as endotoxin. Suitable collagenase preparationsinclude recombinant and non-recombinant collagenase. Non-recombinantcollagenase may be obtained from F. Hoffmann-La Roche Ltd, Indianapolis,Ind. and/or Advance Biofactures Corp., Lynbrook, N.Y. Recombinantcollagenase may also be obtained as disclosed in U.S. Pat. No.6,475,764.

By way of example, in some embodiments, the adipose tissue is treatedwith collagenase solutions with from about 0.5 μg/ml to about 100 μg/ml,e.g., 10 μg/ml to about 50 μg/ml collagenase, and are incubated at fromabout 30° C. to about 38° C. for from about 20 minutes to about 60minutes. These parameters will vary according to the source of thecollagenase enzyme, optimized by empirical studies, in order to validatethat the system is effective at extracting the desired cell populationsin an appropriate time frame. For example, in some embodiments, thetissue is incubated with a solution comprising collagenase for 10-15minutes, at about 37° C.

Following disaggregation the lipo-digestate can be washed/rinsed toremove additives and/or by-products of the disaggregation process, e.g.,collagenase and/or other enzymatic disaggregation agents, andnewly-released free lipid.

In some embodiments, the lipo-digestate can be applied to a portion ofunprocessed, dried, or dehydrated adipose tissue under conditions thatallow the lipo-digestate to permeate through the unprocessed, dried, ordehydrated adipose tissue. For example, in some embodiments, thelipo-digestate can be resuspended, layered over (or under) a portion ofunprocessed adipose tissue, dried adipose tissue or dehydrated adiposetissue and the lipo-digestate is filtered through the unprocessedadipose tissue (or dried or dehydrated adipose tissue) usinggravitational forces. In some embodiments, the lipo-digestate is pumpedthrough the unprocessed adipose tissue, for example using a peristalticpump, vacuum or the like. In some embodiments, the lipo-digestate isadded to the unprocessed adipose tissue to create a mixture, and themixture is agitated or rocked, either mechanically or manually. As thelipo-digestate is filtered through or mixed with the unprocessed adiposetissue, adipose-derived regenerative cells can become bound by theconnective tissue matrix, and saline and other fluids, flow through thetissue, thereby producing a fat graft or implant supplemented orenhanced with adipose-derived regenerative cells (e.g., adipose-derivedregenerative cell comprising stem cells). In some embodiments, theflow-through, e.g., saline, mature adipocytes, red blood cells, and thelike is removed to a waste container. Systems and devices for generatingsupplemented adipose tissue grafts are discussed in more detail below,and one embodiment of the method is depicted in the schematic shown inFIG. 1.

FIG. 1 shows a schematic of an exemplary pathway for preparing asupplemented adipose-tissue graft. In the first step, a unit of adiposetissue is provided into a closed/sterile container (e.g., a collapsible,flexible bag or a rigid container as described elsewhere herein), via aninlet. The adipose tissue is rinsed/washed and digested within thecontainer while maintaining a closed system, as described herein. In theembodiment shown in FIG. 1, the first container has an inlet and anoutlet. The first container shown in FIG. 1 shows a single inlet and asingle outlet, however, the skilled artisan will appreciate that devicesdescribed herein can include multiple, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more inlets and outlets. Preferably, the inlet(s) and theoutlet(s) are configured for aseptic addition and/or removal of contents(e.g., tissue, additives, solutions, and the like) in the firstcontainer.

In the embodiment disclosed in FIG. 1, a second unit of adipose tissue,or portion of the first unit of adipose tissue is provided in a secondcontainer (e.g., a collapsible, flexible bag or a rigid container, asdescribed elsewhere herein). In the embodiment shown in FIG. 1, thesecond container has an inlet, or inlets and an outlet or outlets. Theinlet of the second container is configured for the addition of contents(e.g., lipo-digestate or concentrated populations of adipose-derivedcells) into the container, preferably while maintaining a closed sterilefluid pathway. The outlet is configured for the removal of contents,e.g., excess wash solution, free lipid, blood, and the like from thesecond container.

In the embodiment shown in FIG. 1, following digestion, thelipo-digestate and non-disaggregated adipose tissue fragments, and freelipid form different layers within the first container. Thelipo-digestate layer is allowed to exit (e.g., via a pump or vacuum asshown in FIG. 1) through an outlet in the closed container, and toenter, e.g., through a conduit that maintains the closed system, into aseparate container that contains unprocessed or washed, dried ordehydrated adipose tissue. The lipo-digestate from the first containermixed with the unprocessed, dried or dehydrated adipose tissue underconditions that allow the separated adipose-derived regenerative cellsin the lipo-disgestate or concentrated adipose-derived cell populationto permeate through the adipose tissue. In FIG. 1, the regenerativecells are pumped through the unprocessed or dried adipose tissue tocreate the supplemented adipose tissue graft.

In some embodiments, any excess lipo-digestate or concentratedadipose-derived cell solution is recirculated through the theunprocessed, dried or dehydrated adipose tissue in the second container,by providing an aseptic loop in the second container, wherein the excessregenerative cell solution or lipo-digestate drains through an exit port(outlet) into a conduit that leads to an entry port (inlet) in thecontainer housing the adipose tissue.

It will be appreciated that in making the fortified or supplementedadipose tissue grafts described herein, the volumes of the various unitsor portions of adipose tissue used to produce the lipo-digestate orconcentrated adipose-derived cell solutions and to serve as the base orfoundation for the adipose tissue graft that is supplemented with theadipose-derived regenerative cells or lipo-digestate may be equal, orthey may be different. For example, the volume of adipose tissue used tomake the lipo-digestate can be at least, greater than or equal to about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%,140%, 150%, 160%, 170%, 180%, 190%, 200, 210%, 220%, 230%, 240%, 250%,260%, 270%, 280%, 290%, 300%, or any number in between this range, morethan the volume of another unit of adipose tissue. In some embodiments,the volume of adipose tissue used to make the lipo-digestate can be atleast, greater than or equal to about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%,200, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, or anynumber in between this range, less than the volume of another unit ofadipose tissue. In some embodiments, the ratio of lipo-digestate:grafttissue is about 0.25:1, 0.5:1, 0.75:1, 1:1, 1.25:1, 1.5:1, 1.75:1 or2:1, or any number in between this range. Preferably, the ratio oflipo-digestate: graft tissue less than about 1:1, such as 0.5:1 or0.25:1.

In some embodiments, the portion of processed adipose tissue (e.g.,digested lipoaspirate, or regenerative cell solution), and/or a portionof unprocessed adipose tissue, dried adipose tissue, dehydrated adiposetissue, and/or a adipose tissue graft supplemented with adipose-derivedregenerative cells described herein can be combined, fortified,supplemented, enhanced, or mixed with additives such as other cells,tissue, tissue fragments, demineralized bone, or factors or agents, suchas additives that lyse adipocytes and/or red blood cells. For example,in some embodiments, the portion of processed adipose tissue, and/or aportion of unprocessed adipose tissue (or dried or dehydrated adiposetissue), and/or a adipose tissue graft supplemented with adipose-derivedregenerative cells described herein can be combined, supplemented, ormixed with growth factor additives such as insulin or drugs such asmembers of the thiaglitazone family, antibiotics, biologically active orinert compounds, such as coagulases, cell-reaggregation inhibitors,resorbable plastic scaffolds, or other additive intended to enhance thedelivery, efficacy, tolerability, or function of the population.

In certain embodiments, the unprocessed adipose tissue, the driedadipose tissue, the dehydrated adipose tissue, the lipo-digestate,and/or the supplemented adipose tissue grafts can be supplemented withone or more cellular differentiation agent additives, such as cytokinesand growth factors. In some embodiments, the subject receiving theadipose tissue graft is provided one or more cellular differentiationagents, such as cytokines and growth factors separately, i.e., in adifferent composition, from the adipose tissue graft. For example, insome embodiments, the compositions are supplemented with anigiogenicagents, or factors. In some embodiments, the unprocessed adipose tissue,the dried adipose tissue, the dehydrated adipose tissue, thelipo-digestate, and/or supplemented adipose grafts described herein areprovided an angiogenic factor(s) as an additive. As used herein, theterm “angiogenesis” refers to the process by which new blood vessels aregenerated from existing vasculature and tissue (Folkman, 1995). As usedherein, the term “angiogenic factor” or “angiogenic protein” refers toany known protein, peptide or other agent capable of promoting growth ofnew blood vessels from existing vasculature (“angiogenesis”). Suitableangiogenic factors for use in the invention include, but are not limitedto, Placenta Growth Factor (Luttun et al., 2002), Macrophage ColonyStimulating Factor (Aharinejad et al., 1995), Granulocyte MacrophageColony Stimulating Factor (Buschmann et al., 2003), Vascular EndothelialGrowth Factor (VEGF)-A, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E (Mints etal., 2002), neuropilin (Wang et al., 2003), fibroblast growth factor(FGF)-1, FGF-2(bFGF), FGF-3, FGF4, FGF-5, FGF-6 (Botta et al., 2000),Angiopoietin 1, Angiopoietin 2 (Sundberg et al., 2002), erythropoietin(Ribatti et al., 2003), BMP-2, BMP4, BMP-7 (Carano and Filvaroff, 2003),TGF-beta (Xiong et al., 2002), IGF-1 (Shigematsu et al., 1999),Osteopontin (Asou et al., 2001), Pleiotropin (Beecken et al., 2000),Activin (Lamouille et al., 2002), Endothelin-1 (Bagnato and Spinella,2003) and combinations thereof. Angiogenic factors can actindependently, or in combination with one another. When in combination,angiogenic factors can also act synergistically, whereby the combinedeffect of the factors is greater than the sum of the effects of theindividual factors taken separately. The term “angiogenic factor” or“angiogenic protein” also encompasses functional analogues of suchfactors. Functional analogues include, for example, functional portionsof the factors. Functional analogues also include anti-idiotypicantibodies which bind to the receptors of the factors and, thus, mimicthe activity of the factors in promoting angiogenesis. Methods forgenerating such anti-idiotypic antibodies are well known in the art andare described, for example, in WO 97/23510, the contents of which areexpressly incorporated by reference in its entirety.

Angiogenic factors useful in the embodiments disclosed herein can beproduced or obtained from any suitable source. For example, the factorscan be purified from their native sources, or produced synthetically orby recombinant expression. The factors can be administered to subjectsas a protein composition, in the form of an expression plasmid encodingthe factors, or mixed in with the compositions disclosed herein. Theconstruction of suitable expression plasmids is well known. Suitablevectors for constructing expression plasmids, include, for example,adenoviral vectors, retroviral vectors, adeno-associated viral vectors,RNA vectors, liposomes, cationic lipids, lentiviral vectors andtransposons.

In some embodiments, the cells of the processed adipose tissue, e.g.,lipo-digestate, or regenerative cell solution, the cells of theunprocessed adipose tissue, the dried adipose tissue, the dehydratedadipose tissue, or the cells of the supplemented adipose tissue graftsdescribed herein can also be modified by insertion of DNA or byplacement in cell culture in such a way as to change, enhance, orsupplement the function of the compositions for derivation of acosmetic, structural, or therapeutic purpose. For example, gene transfertechniques for stem cells are known by persons of ordinary skill in theart, as disclosed in Mosca, J. D., J. K. Hendricks, et al. (2000).“Mesenchymal stem cells as vehicles for gene delivery.” Clin Orthop (379Suppl): S71-90, and may include viral transfection techniques, and morespecifically, adeno-associated virus gene transfer techniques, asdisclosed in Walther, W. and U. Stein (2000). “Viral vectors for genetransfer: a review of their use in the treatment of human diseases.”Drugs 60(2): 249-71, and Athanasopoulos, T., S. Fabb, et al. (2000).“Gene therapy vectors based on adeno-associated virus: characteristicsand applications to acquired and inherited diseases (review).” Int J MolMed 6(4): 363-75. Non-viral based techniques may also be performed asdisclosed in Muramatsu, T., A. Nakamura, et al. (1998). “In vivoelectroporation: a powerful and convenient means of nonviral genetransfer to tissues of living animals (Review).” Int J Mol Med 1(1):55-62. In preferred embodiments, the cells of the processed tissue arenot cultured. More preferably, the cells of the processed tissue aremaintained within a closed, sterile system until their use, e.g., untilthey are either loaded onto a delivery device, combined with unprocessedor dried or dehydrated adipose tissue, or delivered directly into asubject.

In embodiments wherein the adipose tissue grafts are administered to apatient other than the patient from which the cells and/or tissue wereobtained, one or more immunosuppressive agent additives may beadministered to the patient receiving the graft to reduce, andpreferably prevent, rejection of the transplant. Examples ofimmunosuppressive agents suitable with the methods disclosed hereininclude agents that inhibit T-cell/B-cell costimulation pathways, suchas agents that interfere with the coupling of T-cells and B-cells viathe CTLA4 and B7 pathways, as disclosed in U.S. patent Pub. No.20020182211. Other examples include cyclosporin, myophenylate mofetil,rapamicin, and anti-thymocyte globulin.

The supplemented adipose tissue graft produced by the methods disclosedherein can be administered directly into the subject. As used herein,the terms “administering,” “introducing,” “delivering,” “placement” and“transplanting” are used interchangeably herein and refer to theplacement of the compositions disclosed herein, e.g., supplemented fatgrafts, into a subject by a method or route which results in at leastpartial localization of the transplant or fat graft at a desired site.In some embodiments, the supplemented adipose tissue graft (e.g., theadipose graft supplemented adipose-derived regenerative cells) can beadministered to the subject without being removed from the system orexposed to the external environment of the system or device in which itwas generated prior to administration. Providing a closed system reducesthe possibility of contamination of the material being administered tothe subject. Thus, processing the adipose tissue and generating thesupplemented adipose tissue graft while maintaining a closed systemprovides advantages over existing methods because the active cellpopulation is more likely to be sterile. In such an embodiment, the onlytime the lipo-digestate or the supplemented adipose tissue graft areexposed to the external environment, or removed from the system, is whenthe cells or supplemented grafts are being withdrawn into an applicationdevice and being administered to the patient. In one embodiment, theapplication device can also be part of the closed system. Thus, in someembodiments, the lipo-digestate or supplemented adipose tissue graftsare not processed for culturing, or cryopreserved.

In some embodiments, at least a portion of the unprocessed tissue, thedried adipose tissue, the dehydrated adipose tissue, the lipo-digestate,and/or supplemented adipose tissue graft is stored for laterimplantation/infusion. For example, the compositions disclosed herein(i.e., the unprocessed tissue, dried adipose tissue, dehydrated adiposetissue, lipo-digestate, concentrated cell adipose-derived cellpopulations and/or supplemented adipose tissue graft) can be dividedinto more than one aliquot or unit such that part of the composition isretained for later application while part is applied immediately to thepatient.

At the end of processing, the supplemented or fortified adipose tissuegraft can be loaded into a delivery device, such as a syringe, scaffold,absorbable capsule or implant, for placement into the recipient by, forexample subcutaneous techniques. In other words, the supplemented,enhanced, or fortified fat graft or implant may be placed into thepatient by any means known to persons of ordinary skill in the art, forexample, they may be introduced into the dermis (subcutaneous), intotissue space, or into tissues (e.g., breast, buttocks, or the like), orother location. Preferably, the loading takes place while maintaining aclosed system. Preferred embodiments include placement by needle,catheter, or by direct surgical implantation in association withadditives, such as a preformed matrix or absorbable breast-shapedcapsules.

As used herein, the term “subject” includes warm-blooded animals,preferably mammals, including humans. In a preferred embodiment, thesubject is a primate. In an even more preferred embodiment, the subjectis a human.

Devices for Producing Dried Adipose Tissue Grafts and SupplementedAdipose Tissue Grafts

As discussed above, provided herein are devices and/or systems formaking dried or dehydrated adipose tissue, and supplemented or fortifiedadipose tissue grafts. Turning to FIG. 2, shown is an exemplary systemfor the production of adipose tissue grafts suitable for supplementationwith adipose-derived regenerative cells. The system comprises a flexiblecollection container 200, e.g., a medical grade collection bag, with alayer of filter mesh 210 that partitions the bag into a first 280 and asecond internal chamber 290. In some embodiments, the filter cancomprise a plurality of openings or pores that permit the passage ofcontents, e.g., mature adipocytes, red blood cells, saline, and thelike, from the first internal chamber into the second internal chamber.Preferably the plurality of pores in the filter are greater than about30 μm. For example, in some embodiments, the plurality of openings inthe filter can be about greater than, less than, or equal to 30 μm, 35μm, 40 μm, 50 μm 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220μm, 230 μm, 240 μm, 250 μm, 260 μm, 270 μm, 280 μm, 290 μm, and 300 μm,or any number in between this range. For example, in some embodiments,the plurality of openings in the filter 210 can be between about 30 μmto about 500 μm. (e.g., about 60 μm-300 μm, such as 74 to about 265 μm).

In some embodiments, the first internal chamber 280 includes two ports220, 230. In some embodiments, the second internal chamber 290 includesone port, 240. In some embodiments, the flexible collection container200 comprises a wash container 250 for washing or rinsing solution,which is operably coupled to internal collection chamber 280 via port220, to enable passage of solution from container 250 to internalchamber 280 through a closed fluid pathway. In some embodiments, port240 is operably coupled to a waste bag 260, through which contents, suchas red blood cells, saline, mature adipocytes and the like are removedfrom internal chamber 290.

In some embodiments, adipose tissue is added to the flexible collectionchamber 200 though port 220. In some embodiments, a rinsing solution,such as Lactated Ringers solution, is added to the first internalchamber through port 230. The flexible collection container is thenagitated or rocked, e.g., on a mechanical rocker, or other agitationdevice. Red blood cells, excess rinsing solution, lysed cells, andmature adipocytes and lipid are removed from the tissue present in thechamber 280.

In some embodiments, the system is configured to allow the asepticaddition of lipo-digestate to internal chamber 280, housing the rinsedadipose tissue. For example, the flexible collection container 200 maycontain an additional port providing a sterile entry pathway intointernal chamber 280, through which lipo-digestate can be directed.

FIGS. 3-13 show additional embodiments of the systems disclosed hereinfor the production of optimized adipose tissue grafts. FIGS. 3 and 4show an exemplary configuration of a system 300, which provides aclosed, sterile process for controlling the hydration of an adiposetissue graft, e.g., to crate dried or dehydrated adipose tissue. Forexample, a common problem associated with preparing adipose tissueimplants concerns the unpredictability of the behavior of the graftafter implantation due to resorbtion or absorption of fluids from thegrafted tissue into the body. As discussed below, the system 300provides the operator with the ability to reduce this variability bycreating a drier graft than that of conventionally obtained adiposetissue. Further, this drier adipose tissue graft or implant can be,optionally, supplemented with lipo-digestate (or concentratedadipose-derived cell populations comprising regenerative cells), oradministered directly to a subject.

FIGS. 3 and 4 show the configuration of one embodiment of system 300 forthe preparation of dried or dehydrated adipose tissue. System 300comprises a first outer shell 310 and a second outer shell 340(collectively and interchangeably referred to herein as “outer shells”or “outer shell”) sealed together to form the outer layer of system 300.FIG. 5 shows an exemplary outer shell 310 (or 340) used in system 300.As discussed below, the outer shells 310, 340 can be affixed, joined, orsealed together to form a flexible, collapsible bag. FIG. 8 shows adetail of an exemplary seal 311 that can be used in the manufacture ofsystem 300. The first and second outer shells 310 and 340 may be madefrom any medical grade flexible material known in the art, e.g., medicalgrade USP Class VI or Medical Grade ethyl vinyl acetate (EVA). In someembodiments, the flexible material that forms the first and second outershells may be made of any material that can be bonded to itself. Inother embodiments, the flexible material that forms the first and secondouter shells may be made of any material that can be bonded to itselfand be able to capture and/or seal any other material present in thesystem or subsystems. The outer shells may also be made from materialthat can withstand cryopreservation. The outer shells may also be madefrom material that is autoclavable, materials that are clear or colored,materials that are biocompatible, materials that are resistant to bodyfluids and/or materials that are sterilizable, e.g, with radiation,ethylene oxide, or dry heat.

By way of example only, the material may be Medical Grade EVA. Inpreferred embodiments, the material is one that can be bonded to itselfand capture other material, e.g., filters present within the system. Thebonding can be accomplished by processes further described herein suchas RF welding. In certain embodiments, the outer shells may be sealedtogether by a double heat seal along the perimeter of the outer shellsas in system 300 shown in FIGS. 3-13. The outer shells or any of thesubsystems may be sealed using adhesives known to one of skill in theart. Types of adhesives to be used, including their mechanism of actioncould be considered. For example, adhesives which harden by loss ofsolvent, adhesives which harden by loss of water, adhesives which hardenby cooling, adhesives which harden by chemical reaction, adhesives whichdo not harden—pressure sensitive adhesives may be used. Mechanisms suchas adhesion by physical adsorption, adhesion by chemical bonding,electrostatic theory of adhesion, mechanical interlocking, adhesion byinterdiffusion, weak boundary layers, and pressure sensitive adhesionmay be considered. The surfaces to be joined must also be addressed suchas surface topography, surface thermodynamics, and surface chemicalanalysis. Certain surfaces to be joined may also require particularpre-treatments for optimum sealing. For example, appropriatepretreatments for metals, pretreatments for inorganic materials,pretreatments for plastics and pretreatments for elastomers may beconsidered.

In some embodiments, the systems and subsystems created advantageouslypossess mechanical properties that allow them to withstand stress. Thus,a global stress analysis, as well as, finite element analysis ofadhesive joints must be performed. The durability of the adhesive jointsmust also be assessed. For example, additives to reduce photo-oxidativedegradation must be considered. Behavior of structural joints to metalsin wet surroundings must be considered. Water and adhesives, water andadhesive interfaces, other fluids, and timber joints must also beconsidered. In some embodiments, nondestructive testing may need to beperformed using conventional ultrasonics, bond testers, rapid scanningmethods and cohesive property measurement. The impact behavior ofadhesively bonded joints may also need to be assessed. For example, animpact test of adhesives and adhesively bonded joints, characteristicsof adhesives under high rate loading and stress distribution andvariation in adhesively bonded joints subject to impact load may beassessed. It may also be desirable to assess fracture mechanics ofadhesive bonds. For example, energy criterion for failure, stressintensity, energy release rate, thermodynamic, intrinsic, and practicaladhesion energy, evaluation of fracture energy and durability. Otherfactors that may be evaluated include fatigue, vibration damping,joining similar and dissimilar materials and bonding composites.

In a particular embodiment, the outer shells of the system, or any ofthe subsystems, or any suitable combination may be sealed using RadioFrequency (RF) welding. RF welding is also referred to as Dielectric orHigh Frequency (HF) welding. RF welding is the process of applying radiofrequency to fuse materials together. The resulting weld can be asstrong as the original materials. RF welding relies on certainproperties of the material being welded to cause the generation of heatin a rapidly alternating electric field. Specifically, the processinvolves subjecting the parts to be joined to a high frequency (13-100MHz) electromagnetic field applied between two metal bars which causesheating of the material to be fused together. Only certain materials canbe welded using this technique. Polyvinylchloride (PVC) andpolyurethanes are the most common thermoplastics to be welded by the RFprocess. It is possible to RF weld other polymers including nylon, PET,EVA and some ABS resins, although special conditions may be required.For example, nylon and PET are weldable if preheated welding bars areused in addition to the RF power. RF welding may not be suitable forPTFE, polycarbonate, polystyrene, polyethylene or polypropylene.However, a special grade of polyolefin has been developed which doeshave the capability to be RF welded.

The primary function of RF welding is to form a joint in two or morethicknesses of sheet material. By incorporating a cutting edge adjacentto the welding surface, the process can simultaneously weld and cut amaterial. The cutting edge compresses the hot plastic sufficiently toallow the excess scrap material to be torn off, hence this process isoften referred to as tear-seal welding. It is also possible to weldadditional pieces of material onto the surface of a product.

In some embodiments, the system and subsystems may also be sealed usingultrasonic welding. When bonding material through ultrasonic welding,the energy required comes in the form of mechanical vibrations. Thewelding tool (sonotrode) couples to the part to be welded and moves itin longitudinal direction. The part to be welded on remains static. Theparts to be bonded are simultaneously pressed together. The simultaneousaction of static and dynamic forces causes a fusion of the parts withouthaving to use additional material. This procedure can be used on anindustrial scale for linking both plastics and metals that may be usedin the system described herein. For ultrasonic welding of plastics, thethermal rise in the bonding area is produced by the absorption ofmechanical vibrations, the reflection of the vibrations in theconnecting area, and the friction of the surfaces of the parts. Thevibrations are introduced vertically. In the contraction area,frictional heat is produced so that material plasticizes locally,forging an insoluble connection between both parts within a very shortperiod of time. The prerequisite is that both working pieces have a nearequivalent melting point. The joint quality in ultrasonic welding isvery uniform because the energy transfer and the released internal heatremains constant and is limited to the joining area. In order to obtainan optimum result, the joining areas are prepared to make them suitablefor ultrasonic bonding. Besides plastics welding, ultrasonics can alsobe used to rivet working parts of the system described herein or embedmetal parts into plastic as needed.

The skilled artisan will appreciate that the systems disclosed hereincan be made from materials other than the flexibile materials discussedabove. For example, in some embodiments, the system or components of thesystem described herein can be manufactured from metals. In suchembodiments, ultrasonic metal welding may be used to join, or affixsystem components to each other. Unlike in other processes, the parts tobe welded are not heated to melting point, but are connected by applyingpressure and high-frequency mechanical vibrations. In contrast toplastics welding, the mechanical vibrations used during ultrasonic metalwelding are introduced horizontally. Specifically, during ultrasonicmetal welding, a complex process is triggered involving static forces,oscillating shearing forces and a moderate temperature increase in thewelding area. The magnitude of these factors depends on the thickness ofthe workpieces, their surface structure, and their mechanicalproperties. The workpieces are placed between a fixed machine part, i.e.the anvil, and the sonotrode, which oscillates horizontally during thewelding process at high frequency (usually 20 or 35 or 40 kHz). The mostcommonly used frequency of oscillation (working frequency) is 20 kHz.This frequency is above that audible to the human ear and also permitsthe best possible use of energy. For welding processes which requireonly a small amount of energy, a working frequency of 35 or 40 kHz maybe used.

The exemplary system 300 shown in FIGS. 3-13 comprises a first andsecond subsystem or first and second chambers created by inserting afilter material 320 between outer shell 310 and outer shell 340. Thefirst subsystem or chamber is defined by the area between outer shell310 and filter material 320 and the second subsystem or chamber isdefined by the area between filter material 320 and outer shell 340. Incertain embodiments, a double heat seal along the perimeter of system300 captures the filter material 320 such that two distinct subsystemsor chambers are formed within the system 300. The filter material cancomprise a plurality of openings that would ideally enable or allow forthe passage of a majority of certain contents, e.g., liquids, tumescentfluids, red blood cells, wash solutions (e.g., saline, lactated ringerssolution, and the like), cellular debris and retention of a majority ofcertain contents e.g., mature adipocytes, regenerative cells, stemcells, progenitor cells and connective tissue. The components that passthrough and those that are retained will be determined by the size ofthe openings in the filter material, i.e., generally components smallerthan the openings will pass through and components larger than theopenings will be retained. The filter material may accordingly beselected based on the size of the components of interest. It is to beunderstood that depending upon the conditions in the system, e.g.,pressure, air flow, viscosity, etc, the number or percentage ofcomponents smaller than the openings on the filter material that willpass through and the number or percentage of components larger than theopenings that will be retained, may differ.

The filter material preferably has a plurality of pores allowing fluidcommunication within or between subsystems. The pores enablecompositions (or components thereof) inserted in the one subsystem todiffuse into another subsystem or vice versa. The pores are preferablylocated on a substantial area of the surface of any filter that may beused. An exemplary filter is shown in FIG. 6.

Any filter that allows excess liquids, red blood cells, or cellulardebris may be used in system 300. For example, any filter that retainsadipocytes, regenerative and stem cells, or connective tissue may beused. Some embodiments provide a system 300 in which the pores of thefilter material 320 can be range from about 1 micron to about 750microns, e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690,700, 710, 720, 730, 740, or 750 microns, or any number or range inbetween. Preferably, the plurality of openings or pores in the filtermaterial is greater than 40 μm. For example, in one embodiment, theplurality of openings in the filter material 320 can be 74 microns. Inother embodiments, the plurality of openings can range from about 73 toabout 264 microns.

As shown in FIGS. 3-13, in some embodiments, the second subsystem orchamber, i.e., the area between filter material 320 and outer shell 340described above can be further divided into two subsystems such that athird subsystem or chamber is formed. For example, a separator 330, e.g.a separation screen, a mesh, or filter is inserted between filtermaterial 320 and outer shell 340. An exemplary separator comprising aseparation screen is shown in FIG. 7. The second and third subsystems orchambers can contain the solutions, effluents, waste, debris and otherunwanted materials from the first subsystem. In certain embodiments, thesecond and third subsystems or chambers are partially distinct from eachother. In some embodiments, the second and third subsystems or chambersare fully distinct from each other. For example, in some embodiments,the separator 330, e.g., separation screen, has a degree of freedom, oris completely free-floating within the second subsystem such that athird subsystem is formed that is not completely separate from thesecond subsystem. In other embodiments, the second and third subsystemsare completely distinct from each other in that the separator, e.g.,separation screen or the like is captured between the outer layers 310and 340 using any of the joining mechanisms known in the art ordescribed herein, e.g., adhesive joining, RF welding, or ultrasonicwelding. The skilled artisan will readily appreciate that severalapproaches can be used to affix the different layers of system 300,e.g., the first and second outer shells, the filter, and the separator,can be selected to ensure optimal seal strength.

In some embodiments, the separator 330 (e.g., a separation screen) isconfigured to minimize contact between the filter material 320 and theouter shell 340. Minimal contact prevents the filter material 320 andthe outer shell 340 from adhering to one another during the processingof the tissue (e.g., if a vacuum develops within the bag). Thus, theseparation screen 330 creates a space between the filter material 320and the outer shell 340. In certain embodiments the separator cancomprise ribs, struts and other features that create a space between theouter shell 340 and the filter 320. In other embodiments, the side ofthe outer shell 340 that faces filter material 320 can be textured tocreate the requisite space, thereby obviating the need for a distinctseparator. The creation of space between the filter and the outer shellof the system generates a force in the bag that pulls, draws or wicksexcess fluid from the adipose tissue into the second and/or thirdsubsystem. The excess fluid can then be directed into a waste container.The space created by the separator, e.g., separation screen 330, and/orthe material used to create the separator is also preferably designed,configured, or selected to wick the lipid present in the adipose tissuethereby helping to remove the lipid and fluids from the tissue withinthe first subsystem or chamber and further drying or dehydrating thetissue.

In certain embodiments, the separator 330 is made from a porous materialand/or comprises a plurality of openings or pores. In certainembodiments, the plurality of pores in the separator 330 can be about300 to about 3000 microns or any number in between this range, e.g.about 500 to about 2000 microns. Preferably, the plurality of openingsor pores have a diameter that is greater than or equal to about 300microns, 400 microns, 500 microns, 600 microns, 700 microns, 800microns, 900 microns, 1000 microns, 1100 microns, 1200 microns, 1300microns, 1400 microns, 1400 microns, 1500 microns, 1600 microns, 1700microns, 1800 microns, 1900 microns, 2000 microns, 2100 microns, 2200microns, 2300 microns, 2400 microns, 2500 microns, 2600 microns, 2700microns, 2800 microns, 2900 microns, 3000 microns, or any number inbetween this range. In one embodiment, the plurality of openings isabout 1000 microns. In some embodiments, the separator is made from aporous material having an open area that is greater than or equal toabout 10%, 12%, 14%, 16%, 1%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 35%,36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%,65%, or any % within this range. The skilled artisan will appreciatethat larger-sized pores allow faster transfer or drainage of materialfrom the first chamber into the second chamber, and that the pore sizeof the separator can be adjusted to balance wicking and/or drainage orfiltration properties. In some embodiments, liquid and lipid adsorbs toand or fills the open areas of the porous separator. Applicants havemade the surprising discovery that in some embodiments, separators thatcomprise pores larger than the pores in the filter material facilitatethe removal of fluid from the adipose tissue and fluids from the firstchamber of the system. Thus, in some embodiments, the separator, e.g.,the separation screen 330, is a porous material, wherein the pores havea larger diameter or sizes than the filter material 320 described above.

In some embodiments, the separator comprises a biocompatible materialthat traps or wicks lipids and/or fluids. For example, the separatorscan be made of a polyester, a nylon, rayon, cellulose nitrate andcellulose acetate. In some embodiments, the separator is made of aflexible material, and in some embodiments, the separator made of arigid material. Preferably, the separator is made of a polyester mesh,e.g., with a pore size of 1000 microns.

As shown in FIGS. 3-13, the system 300 can comprise one or more ports toallow for adding or removing materials into and out of the system. Forexample, the system 300 shown in FIGS. 3, 4, 8 and 9 have three separateports. Ports 400 and 500 are in communication with the first subsystem,e.g., the area between outer shell 310 and filter material 320. Port 600is in communication with the second and/or the third subsystem, e.g.,the area between the filter material 320 and the separation screen 330and/or the area between separation screen 330 and outer shell 340. Insome embodiments, however, the system can include only one port, or onlytwo ports, e.g., one inlet port and one outlet port. In otherembodiments, the system can include more than three ports, e.g., 4, 5,6, 7, 8, 9, 10 or more ports.

Generally, the ports comprise at least one aperture that extends fromthe environment into the interior of the system or subsystem or viceversa. The apertures have an airtight and watertight seal along theseams of the ports. As discussed further below, in some embodiments, theports are configured to be coupled to one or more connectors, conduits,port assemblies, adaptors, caps, or syringes.

The ports may provide an access point for inserting various fluids,e.g., washing and rinsing fluids, and removing such fluids and effluent.Preferably, the ports can be sealed (e.g., with a valve). In someembodiments, the ports can be manually sealed with a clamp. In someembodiments, the ports can be sealed by a cap. In some embodiments, theports comprise self-sealing valves, e.g., a deformable valve thatprovides for unidirectional flow of fluid or contents into, but not outof the internal chamber(s) of the system. In other embodiments, thedeformable valves provide for bi-directional flow. Deformable valves canbe made from any deformable material known in the art, such as rubber,neoprene, silicone, polyurethane, or the like. In some embodiments, theports comprise a luer-activated valve. In some embodiments, the portsare located in the system such that when the system is held upright, oneor more ports is positioned inferiorly such that fluids and effluentwill egress with the assistance of gravitational forces, suction orpressure. Other ports may also be utilized with the system 300 of theinvention, e.g, ports for venting, ports for adding materials or gasesto the system or subsystems etc.

Ports on the system or subsystem can be configured to be directly orindirectly (i.e., via an adaptor) interconnected with or coupled to asyringe or catheter used to suction the adipose material from the sourcebody such that the adipose tissue is directly transported into systemanaerobically. Similarly, a cannula or syringe maybe attached to a portfor anaerobic transplantation of the refined tissue. Accordingly, insome embodiments, the ports on the system or subsystem are configured tobe directly or indirectly coupled with disposable or re-usable syringes.For example, in some embodiments, the ports are configured to bedirectly or indirectly coupled with a 1 cc syringe, a 2 cc syringe, a 5cc syringe, a 10 cc syringe, a 20 cc syringe, a 50 cc syringe, a 60 ccsyringe, a 100 cc syringe, a 250 cc syringe, or the like. In someembodiments, the ports are configured to be directly or indirectlycoupled with a syringe having a large bore tip, e.g. a Toomey syringe.

In some embodiments, the system 300 comprises a tissue access portassembly 700 to facilitate aseptic delivery or removal of contents(e.g., adipose tissue), to and from the first chamber or subsystem.(See, e.g., FIG. 9). An exemplary tissue access port assembly 700 isshown in greater detail in FIGS. 10 and 11. In some embodiments, thetissue access port assembly comprises a large bore cylindrical opening710. The large size of bore 710 facilitates delivery and removal oftissue into the first chamber. A deformable plastic valve 720 canprovide a barrier that prevents material from passing through theopening 710 into the body of the port 730. The deformable plastic valve720 can be configured to open and permit flow of contents through thebore into the body 730 of the tissue access port, leading into the firstchamber of the system by insertion of a syringe tip (not shown) therein.The deformable plastic valve 720 returns to a closed default state uponremoval of the syringe tip from the valve, thereby sealing the port andblocking the flow of any content (e.g., tissue or liquid), out of thebody of the tissue access port when not in use. In some embodiments, thetissue access port assembly is configured to be connected a cap 740 toprovide an additional seal to the tissue access port when the port isnot in use. A more detailed illustration of an exemplary cap 740 isshown in FIG. 12. In some embodiments, the tissue access port assemblycan be removable from system 300. In some embodiments, the tissue accessport assembly is affixed to or joined to system 300, e.g. via anadhesive such as UV adhesive.

In some embodiments, the tissue access port assembly can be configuredto interconnect with an adaptor or connector. In some embodiments, theadaptor or connector is configured to join a syringe tip to a port ofthe system. As discussed above, by way of example, adaptors such astissue port assemblies can be integral to, or removable from theport(s). In some embodiments, the adaptor or connector comprises a luerconnector. In some embodiments, the adaptor or connector comprises aremoval adaptor comprising a deformable valve.

In some embodiments, ports of the system can comprise, or be connectedto, an assembly configured for more than one flow path, e.g, a Yconnector or the like. In some embodiments, the Y connector isconfigured to allow obstruction of one or both flow paths at as desired,e.g., by clamping the individual lumens or flowpaths of the Y connector.In some embodiments, the Y connector comprises a switch located at thejunction of the common and individual flowpaths of the Y connector thatthat can be adjusted to enable simulataneous flow through eachindividual flowpath, one flowpath, or to obstruct both flowpaths, asdesired.

In some embodiments, the ports of the system can be connected toconduits or tubing that enable fluid communication to one or moresystems or subsystems while maintaining a closed pathway. For example,in some embodiments, the system 300 may optionally comprise a waste bag(not shown), waste receptacle (not shown), waste conduit (not shown)and/or a waste container (not shown). In certain of these embodiments,all of the subsystems are in fluid communication with each other. Inother embodiments, none of the subsystems are in fluid communicationwith each other. In a particular embodiment, one subsystem is sealed offfrom the other two subsystems wherein the two subsystems are in fluidcommunication with each other. Preferably, the entire system, includingthe subsystems, are flexible allowing for the system to be of any sizeor shape and accommodate a wide range of volumes. Alternatively, theentire system, subsystems and associated components, may be rigid. Or,some subsystems and associated components may be flexible whereas othersubsystems and associated components may be rigid.

FIG. 13 illustrates an exemplary embodiment of a system 800, comprisinga system 300-1 in fluid connection with other systems and subsystems. Asshown in FIG. 13, ports 400-1 and 500-1 can connect to a wash solutionsource 810-1 and a waste bag 820-1, respectively. The system 300-1 canbe connected with a similar system 300-2, while maintaining a closedpathway. Adipose tissue is introduced into the first chambers systems300-1 and 300-2 as described herein above, e.g., through tissueentry/removal ports 600-1 and 600-2, respectively. The adipose tissue insystem 300-2 is used to produce a dried or dehydrated graft as describedabove. The adipose tissue in system 300-1 is processed to producelipo-digestate or a concentrated population of adipose-derived cellscomprising regenerative cells. Port 500-1 or an alternative port (notshown) can be connected to a solution source, e.g. 810-1, configured forthe aseptic introduction of wash solution and/or enzymes into the secondchamber of system 300-1 for tissue rinsing and digestion. Port 400-1 canbe connected to a waste bag 820-1. The lipo-digestate in the firstchamber of 300-1 can be aseptically transferred to the dried ordehydrated adipose tissue within the first chamber of system 300-2 via aconduit (not shown) that connects the ports 600-1 and 600-2, whilemaintaining a closed system.

The systems disclosed herein can yield adipose tissue grafts andimplants that have significantly lower amounts of undesirable material,such as red blood cells, white blood cells and lipid, than equivalentamounts of adipose tissue from the same subject at the same site thathave been processed by methods such as centrifugation and/orconventional gravity separation. In some embodiments, the adipose tissuegrafts produced using the systems disclosed herein have at least about1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10× (or any number in between thisrange) fewer white blood cells than that present in an equivalent unitof adipose tissue that has been excised from the same subject at thesame site and prepared using a centrifugation method, e.g., wherein theexcised tissue is spun in a fixed angle centrifuge. For example, in someembodiments, the adipose tissue grafts produced in the systems disclosedherein contain less than 75%, less than 80%, less than 85%, less than90%, less than 95%, or less, or any % in between, of the number of whiteblood cells in an equivalent unit of adipose tissue from the sameindividual.

In some embodiments, the adipose tissue grafts produced using thesystems disclosed herein have at least about 1×, 2×, 3×, 4×, 5×, 6×, 7×,8×, 9×, 10× (or any number in between this range) fewer red blood cellsthan in an equivalent unit of adipose tissue that has been excised fromthe same subject at the same site and prepared using a centrifugationmethod, e.g., wherein the excised tissue is spun in a fixed anglecentrifuge. For example, in some embodiments, the adipose tissue graftsproduced in the systems disclosed herein contain less than 75%, lessthan 80%, less than 85%, less than 90%, less than 95%, or less, or any %in between, of the number of red blood cells in an equivalent unit ofadipose tissue.

In some embodiments, the adipose tissue grafts produced using thesystems disclosed herein have at least about 1×, 2×, 3×, 4×, 5×, 6×, 7×,8×, 9×, 10× (or any number in between this range) less lipid than in anequivalent unit of adipose tissue that has been excised from the samesubject and prepared using a centrifugation method, e.g., wherein theexcised tissue is spun in a fixed angle centrifuge. For example, in someembodiments, the adipose tissue grafts produced in the systems disclosedherein contain less than 75%, less than 80%, less than 85%, less than90%, less than 95%, or less, or any % in between, of the percentage oflipid content than in an equivalent unit of adipose tissue.

In addition to the ability to produce adipose tissue grafts with fewerundesirable components, the grafts produced by the systems disclosedherein can also exhibit hydration characteristics that facilitatesupplementation with adipose-derived regenerative cells and/or retentionof the implant. Specifically, the hydration state of an adipose tissuegraft prepared as described herein is less hydrated than a graft of anequivalent unit of adipose tissue isolated from the same subject at thesame site and prepared using gravity separation alone.

The skilled artisan will appreciate, however, that in some embodiments,systems can be designed with a flexible bag that is not comprised of twodifferent outer shells sealed together, but that is, rather, a seamlessbag. A filter can be sealed, joined or affixed within the seamless bag,to define first and second subsystems or chambers within the interior ofthe bag.

An exemplary device 100 for producing the supplemented, enhanced, orfortified fat grafts or adipose tissue implants disclosed herein isshown in FIG. 14. FIG. 14A shows a perspective view of a canister 10that includes at least two chambers 20, 30. One chamber 20 can be usedto produce lipo-digestate from unprocessed tissue. A second chamber 30can be used to receive and store unprocessed adipose tissue, to be usedas the fat graft in the generation of the supplemented adipose tissuegraft.

In the embodiment shown in FIG. 14, each chamber 20, 30, has a tissueentry port 40, 50, configured to receive unprocessed adipose tissue. Itwill be appreciated, however, that in some embodiments, the canister 10has only one tissue entry port. In some embodiments, the tissue entryports 40, 50 are configured to operably connect to a cannula (notshown), such that lipoaspirate directly enters into the chamber(s) 20,30 during liposuction. For example, the tissue inlet port 40, 50 can becoupled to a cannula (not shown) by way of tubing to define a tissueremoval line. In some embodiments, the cannula can be an integrated,single-use liposuction cannula, and the tubing can be a flexible tubing.The cannula can dimensioned to be inserted into a subject to removeadipose tissue from the subject. The tubing used in the system can becapable of withstanding negative pressure associated with suctionassisted lipoplasty to reduce the likelihood of collapsing. A suctiondevice (not shown) such as a syringe or electric vacuum, among otherthings, can be coupled to the canister 10, and configured to provide asufficient negative pressure to aspirate tissue from a subject.

The chambers 20, 30 of the canister 10 can be physically separated fromeach other, such that the flow of contents of one chamber 20 (e.g., thechamber containing lipo-digestate) into the other chamber 30 (e.g., thechamber containing the fat graft), is controlled. In some embodiments,the chambers are in fluid connection, for example, through a port thatcan be sealed off, to ensure flow of contents from one chamber to theother chamber only when desired. For example, in some embodiments, flowof contents from one chamber 20 to another chamber 30, occurs through aconduit. The optional conduit can include one or more clamps (not shown)to control the flow of material among various components of the system.The clamps can be used to maintain the sterility of the system byeffectively sealing different regions of the system. Alternatively, theoptional conduits may include one or more valves that control the flowof material through the system. The valves can be electromechanicalpinch valves, pneumatic valves, hydraulic valves or mechanical valves.In some embodiments, the valves can be activated or actuated by acontrol system, which may be coupled to levers. The levers can bemanually manipulated and/or automatically manipulated, for example,through a processing device which may activate the valves atpredetermined activation conditions. In certain automated embodiments,activation of the valves may be partially automated and partiallysubject to the user's preference such that the process may be optimized.In yet other embodiments, certain valves may be activated manually andothers automatically through a processing device. The valves may also beused in conjunction with one or more pumps, e.g., peristaltic pumps orpositive displacement pumps (not shown). The conduits and/or the valvescan also include sensors, such as optical sensors, ultrasonic sensors,pressure sensors or the like, that are capable of distinguishing amongthe various fluid components and fluid levels that flow through thesystem.

In some embodiments, one or both chambers can include one or more ports60 for the removal of waste, e.g., saline, mature adipocytes, red bloodcells, and the like, the addition of components, e.g., wash solution,enzymes, cells, and the like, or for an air inlet or outlet vent. Insome embodiments, chamber 30 can include a port, or outlet 70 configuredfor the aseptic removal of the supplemented fat graft. Outlet 70 can bestructured to pass the composition (e.g., supplemented fat graft) fromchamber 30 to a subject under the appropriate conditions. For example,in some embodiments a syringe can be used to withdraw the composition,and outlet 70 is able to accommodate a needle of the syringe withoutcompromising the sterility of the system or composition. In additionalembodiments, the outlet can be coupled to a device that is configured toadminister the composition, but not to withdraw the composition, such asa cannula that administers the composition by applying positive pressureto displace the composition through the cannula. Accordingly, outlet 70can be configured to allow the composition contained in chamber 30 to bepassed into the cannula (not shown). In other embodiments, outlet 70 cancomprise, or be coupled in a closed-system fashion to, the device foradministering the composition, such as a needle of a syringe or acannula for administering the composition by applying positive pressure.

In some embodiments, one or both chambers 20, 30 of the canister 10 canbe configured to aseptically receive solutions and agents, such aswashing solutions (saline, and the like), disaggregation agents, orother agents or additives. The device can include containers configuredto hold their contents in a sterile manner, e.g., a collapsible bag,such as an IV bag used in clinical settings. These containers may haveconduits coupled to one or both chambers 20, 30. For example, the devicecan be configured such that a container holding a rinsing or washingagent (e.g., PBS, PLASMALYTE®, NORMOSO®, or Lactated Ringer's solution)can be aseptically delivered to chamber 20 and chamber 30. In someembodiments, the device 100 can be configured such that a containerholding a disaggregation agent coupled to canister 10 to deliver thedisaggregation agent(s) to the interior of chamber 20. Solutions andagents can be delivered to the interior of the chambers 20, 30 of thecanister through any art-recognized manner, including gravity pressureapplied to the outside of the containers, or by placement of a positivedisplacement pump on the conduits. In automated embodiments, aprocessing device calculates various parameters, e.g., the volume ofsaline and time or number of cycles required for washing, as well as,the concentration or amount of disaggregation agent and the timerequired for disaggregation based on information initially entered bythe user (e.g., volume of tissue being processed). Alternatively, theamounts, times etc. can be manually manipulated by the user. In someembodiments, the device is configured to agitate, or shake, one or bothchambers of the canister. For example, in some embodiments, the devicecomprises an orbital motion platform 80 configured to agitate thecontents of chamber 20, during the disaggregation process.

The components of the canister 10 can be made of materials that arenon-reactive with biological fluids or tissues, and non-reactive withagents used in processing biological fluids and tissues. In addition,the materials from which the various components are made should becapable of withstanding sterilization, such as by autoclaving, andirradiation, including but not limited to beta- or gamma-irradiation. Insome embodiments, the canister is made from disposable materials. Insome embodiments, the canister can be made from non-disposable material,which can be used more than one time. By way of example, the tubing andthe cannula handle may be made of any suitable material, such aspolyethylene. The cannula may be made of stainless steel. For example,in some embodiments, the canister is made from polycarbonate acrylic,ABS, ethylene vinyl acetate, or styrene-butadiene copolymers (SBC). Thefluid pathway of the device is preferably pyrogen free, i.e., suitablefor blood use without danger of disease transmittal. In someembodiments, the canister 10 is constructed of a material that allowsthe user to visually determine the approximate volume of tissue presentin the chamber.

In some embodiments, the device includes one or more temperature controldevices (not shown) that are positioned to adjust the temperature of thematerial contained within one or more chambers 20, 30 of the system. Thetemperature control device can be a heater, a cooler or both, i.e., itmay be able to switch between a heater and a cooler. The temperaturedevice can adjust the temperature of any of the material passing throughthe device 100, including the tissue, the disaggregation agents,resuspension agents, the rinsing agents, the washing agents oradditives. For example, heating of adipose tissue facilitatesdisaggregation whereas the cooling of the regenerative cell output isdesirable to maintain viability. Also, if pre-warmed reagents are neededfor optimal tissue processing, the role of the temperature device wouldbe to maintain the pre-determined temperature rather than to increase ordecrease the temperature.

In some embodiments, the device 100 can be automated. In someembodiments, the device 100 can include a processing device (e.g.,microprocessor or personal computer) and associated software programsthat provide the control logic for the system to operate and to automateone or more steps of the process based on user input. In certainembodiments, one or more aspects of the system may be user-programmablevia software residing in the processing device. The processing devicemay have one or more pre-programmed software programs in Read OnlyMemory (ROM). For example, the processing device may have pre-programmedsoftware tailored for processing blood, another program for processingadipose tissue to obtain small volumes of regenerative cells and anotherprogram for processing adipose tissue to obtain larger volumes ofregenerative cells. The processing device may also have pre-programmedsoftware which provides the user with appropriate parameters to optimizethe process based on the user's input of relevant information such asthe amount of regenerative cells required, and various post-processingmanipulation, etc.

In some embodiments, the software can automate steps such as controllingthe ingress and egress of fluids and tissues along particular tubingpaths by controlling pumps and valves of the system; controlling theproper sequence and/or direction of activation; detecting blockages withpressure sensors; mixing mechanisms, measuring the amount of tissueand/or fluid to be moved along a particular pathway using volumetricmechanisms; maintaining temperatures of the various components usingheat control devices; and integrating the disaggregation process withtiming and software mechanisms.

The following examples are provided to demonstrate particular situationsand settings in which this technology may be applied and are notintended to restrict the scope of the invention and the claims includedin this disclosure. A number of publications and patents have been citedhereinabove. Each of the cited publications and patents are herebyincorporated by reference in their entireties.

The following example compares the purity and hydration of adiposetissue grafts obtained using the system disclosed herein to equivalentunits of unprocessed adipose tissue, and equivalent units of adiposetissue grafts produced by gravity separation and centrifugation.

EXAMPLE 1

Aspirated adipose tissue was collected from clinical offices by eitherliposuction (N=5), laser (N=3) or Body-jet harvesting (N=2) methods from10 human subjects (N=10). The aspirated tissue samples were randomlydivided into four groups: (1) control; (2); gravity separation; (3)centrifugation; and (4) PUREGRAFT™ tissue graft preparation. The controlsamples were analyzed directly, without further manipulation. Samples inthe gravity separation group were set aside in a 60 mL syringe for tenminutes. The fluid portion of the samples was discarded and theremaining adipose tissue was analyzed. For the centrifugation group,samples were loaded into a capped 10 mL syringe placed into an IEC fixedangle rotor centrifuge at centrifuged at 3000 rpm (˜1,200 g) for 3minutes. Free lipid at the top of the adipose tissue was removed byaspiration and the infranatant drained following centrifugation and theremaining graft tissue was analyzed. Samples in the PUREGRAFT™ groupwere washed in a system 300 described herein above (PureGraft™).Briefly, tissue was introduced into the first chamber of the system 300as described above. The tissue was washed with 2×150 mls of LactatedRinger's solution. The excess fluid and lipid was allowed to drain fromthe second chamber of the system. The dried tissue was removed throughthe tissue entry port and used for result analysis.

The graft tissues from each of the four groups were subsequentlycentrifuged at 400 g for five minutes in 15 ml conical tubes intriplicate in order to separate the grafts into four components; freelipid, adipose tissue, liquid, and a cell pellet comprising red andwhite blood cells. The volumes of lipid layer and liquid layer wererecorded and calculated as percentage of total graft tissue. The volumeof the lipid layer and the liquid layer of each sample was measured, andused to calculate the liquid content and lipid content of the differentpreparations. As shown in FIG. 15, adipose tissue prepared using themethods and systems described herein (PureGraft) had a significantlylower water content, when compared to unprocessed adipose tissue(Control), or adipose tissue processed by a conventional a gravitymethod (Gravity). Specifically, the mean liquid content of tissueprepared using the system of the current invention was 9.3±0.9%. Thiswas significantly lower (p<0.001) than tissue prepared by gravityseparation (25.1±1.8%). As shown in FIG. 16, adipose tissue preparedusing the methods and systems described herein (PureGraft) had asignificantly lower % content (v/v) of lipid compared to unprocessedadipose tissue (Control), adipose tissue prepared by a conventionalgravity method (Gravity) and adipose tissue prepared by a conventionalcentrifugation method (Centrifugation). Specifically, the residual freelipid level in the samples prepared using the system of the currentinvention averaged 0.8±0.3% free lipid compared to control(unmanipulated) tissue (11.5±1.5%, p<0.001), gravity separation(8.9±1.3%, p<0.001), and centrifugation (9.6±1.8%, p<0.001). Note thatthe free lipid content observed in grafts prepared by centrifugation(average 9.6% of graft volume) was measured after removal of the freelipid observed during initial preparation of the graft. That is, thefree lipid evident after separation of the graft into its componentparts is newly released. This indicates that the graft material preparedby centrifugation contained damaged adipocytes that released their lipidduring the second centrifugation applied to separate the graft into itsfour component parts. The fact that application of the same secondcentrifugation step to grafts prepared within system of the presentinvention revealed markedly less free lipid 0.8% of volume compared to9.6%) demonstrates that grafts prepared within the system of the presentinvention contained fewer damaged adipocytes.

To assess the purity of the graft, samples from each tissue graft wereremoved from the tubes and analyzed for blood content by counting redblood cells and white blood cells per gram of tissue with a CoulterCounter. The data were normalized to control groups, and expressed asrelative percentage of either RBC or WBC content per gram of unprocessedgraft tissue. All data were expressed as average±SEM. A student t testwas used to compare the differences between each graft preparationmethod. The data shown in FIG. 17 illustrate that adipose tissueprepared using the methods and systems disclosed herein (PureGraft) havesignificantly less red blood cells (RBCs) per gram of tissue, comparedto unprocessed adipose tissue (Control), adipose tissue prepared by aconventional gravity preparation method (Gravity) and adipose tissueprepared by a conventional centrifugation method (Centrifugation). Thus,while gravity separation and centrifugation removed 47.8±7.0% and53.2±5.4% of red blood cells respectively, preparation using the systemof the present invention removed 98.1±0.01% red blood cells from thegraft. Similarly, white blood cell content was reduced by 58.7±13.7% bygravity, 69.7±5.2% by centrifugation, and 96.80±0.01% using the systemof the present invention.

The presence of water, lipid/mature adipocytes and blood cells can leadto loss of graft volume over time. The data shown in FIGS. 15-17demonstrate that the systems described herein are useful for thepreparation of dried or dehydrated adipose tissue grafts or implants.

The following example describes the production of an adipose tissueimplant or graft supplemented with adipose-derived regenerative cells bythe methods described herein.

EXAMPLE 2

A patient in need of or desiring a breast implant is identified orselected. A unit of adipose tissue is removed from the patient andprovided to an adipose-derived stem cell processing unit, whichpreferably, maintains a closed, sterile fluid/tissue pathway. Forexample, a cannula is connected to a tissue collection container orchamber (e.g., a flexible bag) of the adipose-derived stem cellprocessing unit while maintaining a sterile fluid/tissue pathway.Liposuction is performed by established techniques to remove adiposetissue from the subject and the removed unprocessed adipose tissue isdrawn into the tissue collection container/chamber. A first portion ofthe adipose tissue is rinsed with PBS until substantially all of thesaline, red blood cells, mature adipocytes are removed (e.g., successivewashes until the tissue is no longer visibly red), and the washeffluent—the waste, e.g., saline, red blood cells, mature adipocytes, isaseptically removed through a port that is joined to the tissuecollection container or chamber, while maintaining a closed, sterilefluid/tissue pathway. In some embodiments the waste port is connected toa waste collection container by a conduit and the waste collectioncontainer may be configured to attach to a vacuum source and the conduitand/or waste collection container may contain one or more valves. Thefirst portion of washed adipose tissue can then be stored in the tissuecollection container/chamber or transferred to a storage container orchamber for further processing.

A second portion of the unit of adipose tissue is rinsed with PBS untilthe tissue is no longer visibly red, as described above, in a secondchamber. Alternatively, the adipose tissue obtained by liposuction isrinsed and washed, as described above, and afterward is split into afirst and second portion. The first portion is retained for use as thesubstrate for the addition of adipose-derived regenerative cells and thesecond portion is used to create a suspension of adipose-derivedregenerative cells as follows. An enzyme solution comprising collagenaseis aseptically added to the second portion of tissue while maintaining aclosed sterile fluid/tissue pathway. The tissue is incubated, whilerocking, at 37° C. for approximately 1 hour. The mixture is allowed tosettle, such that the lipo-digestate/adipose derived regenerative cellsolution is physically separated from the undigested adipose tissue andlipid. The lipo-digestate created from the second portion of adiposetissue is removed through a conduit while maintaining a closed, sterilefluid/tissue pathway.

The separated lipo-digestate is then pumped through a conduit that isconnected with the first chamber though a closed pathway. Thelipo-digestate is pumped over and through the first portion ofunprocessed adipose tissue in the first chamber. The non-cellularcomponent of the lipo-digestate flows through the first portion ofunprocessed adipose tissue into a waste container, while maintaining aclosed sterile fluid/tissue pathway. The lipo-digestate can be filteredthrough the first portion of unprocessed adipose tissue using gravity ora vacuum source. In some embodiments, the lipo-digestate can be layeredon top of the first portion of unprocessed adipose tissue and theadipose-derived regenerative cells present in the lipo-digestate can beforced into the matrix of the first portion of unprocessed adiposetissue using centrifugation (e.g., spinning bucket centrifugation at100, 200, 300, 400, 500, 600, 700, or 800 g). The adipose-derivedregenerative cells of the lipo-digestate are bound by the connectivetissue fragments, producing a fat graft supplemented withadipose-derived regenerative cells. The supplemented or fortified fatgraft can then be provided to the subject with or without an absorbablecasing or capsular material that has a shape of a human breast.

EXAMPLE 3

A fat graft supplemented with adipose-derived regenerative cells isprepared as described in Example 1. The fat graft supplemented withadipose-derived regenerative cells is administered to the face,buttocks, chest, or calves of a subject or to correct any soft tissuedefect.

EXAMPLE 4

A patient in need of or desiring a fat graft is identified or selected.A unit of adipose tissue is removed from the patient as described hereinor as known in the art. To optimize the graft, the system 300 isoriented and a waste bag (not shown) connected to the system is droppedto the floor. Adipose tissue is introduced into the system 300 throughtissue access port 600 which provides communication between the externalenvironment and the interior of the first subsystem. A Toomey or otherart recognized syringe may be used to inject the lipoaspirate throughthe port. This may be repeated until the desired amount of tissue hasbeen added—taking care not to exceed the maximum volume of the firstsubsystem. Once the lipoaspirate has been added, the pinch clamp on thedrain tubing (not shown) is closed. A washing and/or rinsing solution,such as Lactated Ringers or saline, is introduced into the system 300 byopening the wheel clamp (not shown) through a port or by way of sterileIV tubing. The amount of washing or rinsing solution to be added mayvary. Generally, sufficient washing solution is introduced into thesystem such that the majority of the lipoaspirate or other tissue in thefirst subsystem is submerged. 150 mls of washing solution such asLactated Ringers is added. Next, the wheel clamp is closed and thesystem is manually agitated for a brief period of time, e.g., 15seconds. The agitation may be in the form of inversion, rotation,rocking, etc. The system is agitated by external means such as on ashaking platform, an orbital shaker etc. After the tissue is thoroughlywashed (as determined by observation or a pre-established timeinterval), a drain pinch valve (not shown) or a port is opened and,under gravitational forces, the effluent passes through the filtermaterial 320 and the separation screen 330. The effluent is allowed todrain for a period of time, e.g., 3 minutes. This cycle can be repeateda number of times. The cycle may be repeated for a total of four washes.After the last wash cycle, the waste should be allowed to drain for fiveto ten minutes. Generally, maximal draining of waste fluids occurs afterabout 10 minutes of draining. Accordingly, a dryer washed fat graft iscreated that is substantially free of blood, tumescent fluid and freelipid without the need for mechanical equipment and in a closed sterilesystem which is easy to operate. Also, this process can be completed inas little as 20 minutes and the surgeon can control the level ofhydration desired by altering the length of time that the system isallowed to drain during the final step.

If enriching the fat graft obtained by using the system 300 with adiposederived regenerative cells (ADRCs) is desired, the ADRCs obtained by anymethod described herein or known in the art (such as by use of theCELUTION® System), may be added via a port near the bag of LactatedRingers and chased with Lactated Ringers or other suitable solution for5 seconds or more. The system can then be agitated as described herein,e.g., for 15 seconds, and the ADRC enhanced fat graft may then beinjected or placed back into the patient as needed.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A device for preparing tissue for an adipose tissue graft,comprising: a flexible, collapsible bag having a first chamber and asecond chamber, which are defined by a filter comprising pores; aseparator located within the second chamber; an inlet port connected tothe flexible, collapsible bag, wherein said inlet port is configured toallow the aseptic introduction of adipose tissue into the first chamber;and an outlet port connected to the flexible, collapsible bag, whereinthe outlet port is configured to aseptically remove liquid and cellsfrom the second chamber.
 2. The device of claim 1, wherein the separatoris a free floating porous structure within said second chamber.
 3. Thedevice of claim 1, wherein the separator is a porous structure thatdefines a third chamber within the second chamber.
 4. The device ofclaim 1, wherein the bag comprises a first outer shell and a secondouter shell joined by a double heat seal.
 5. The device of claim 4,wherein the filter is joined to the first outer shell and the secondouter shell by a double heat seal.
 6. The device of claim 1, wherein theseparator comprises a lipid-wicking material.
 7. The device of claim 6,wherein said lipid-wicking material is a polyester mesh screen.
 8. Thedevice of claim 1, wherein said separator comprises a plurality of poresthat are larger than the pores of said filter.
 9. The device of claim 8,wherein the pore size of said second filter is greater than or equal toabout 5, 6, 7, 8, 9, or 10 times larger than the pore size of saidfilter.
 10. The device of claim 1, wherein the pore size of said filteris greater than or equal to about 30 μM.
 11. The device of claim 1,wherein the pore size of said filter is between about 30 μM and about200 μM.
 12. The device of claim 1, wherein the separator comprises poresand wherein the pore size of said separator is between about 300 and2000 μM.
 13. The device of claim 1, wherein said inlet port isconfigured to releasably connect with an adapter, said adapter beingconfigured to releasably connect with the tip of a syringe barrel. 14.The device of claim 13, wherein the inlet port is configured to allowmaterial to enter into the port, but not to exit from the port.
 15. Thedevice of claim 14, wherein said inlet port comprises a deformableplastic valve.
 16. The device of claim 13, wherein the syringe barrel isa 60 mL catheter syringe barrel.
 17. The device of claim 1, wherein theinlet port is configured to be attached to a cannula, while maintaininga sterile fluid/tissue pathway.
 18. The device of claim 1, furthercomprising a second device, said second device comprising anadipose-derived regenerative cell isolation device attached to saiddevice.
 19. The device of claim 18, wherein said second device comprisesa device of claim
 1. 20. The device of claim 18, wherein the seconddevice is connected to the device of claim 1 by a conduit that isconfigured to transfer isolated adipose-derived regenerative cells fromsaid second device to the first chamber of the device of claim
 1. 21.The device of claim 20, wherein said conduit comprises a Y connection.22. A method of making an adipose tissue graft, comprising: (a)obtaining a first portion of unprocessed adipose tissue; (b) introducingsaid first portion of unprocessed adipose tissue into the first chamberof a first device of claim 1; (c) rinsing the first portion ofunprocessed adipose tissue by adding a physiologic wash solution to thefirst chamber; and (d) removing fluid from the second chamber of thefirst device, wherein the fluid is selected from the group consisting ofwater, physiologic wash solution, blood and free lipid, or anycombination thereof, thereby drying the adipose tissue.
 23. The methodof claim 22, wherein the physiologic solution is selected from the groupconsisting of Lactated Ringer's solution, Hartmann's Solution, saline,and PLASMALYTE™ solution.
 24. The method of claim 22, wherein said driedadipose tissue of (c) is dehydrated to a liquid content that is lessthan about ½, ⅓, or ¼ times that of said first portion unprocessedadipose tissue prior to the introducing step.
 25. The method of claim22, wherein said dried adipose tissue of (c) is dehydrated to a liquidcontent that is less than about ⅓ that of said first portion unprocessedadipose tissue prior to the introducing step.
 26. The method of claim22, further comprising isolating a population of adipose-derivedregenerative cells from a second portion of adipose tissue andcontacting the dehydrated adipose tissue of (d) with the isolatedpopulation of adipose-derived regenerative cells under conditions thatallow the isolated population of adipose-derived regenerative cells topermeate the dried adipose tissue of (d).
 27. The method of claim 26,wherein said isolated population of adipose-derived regenerative cellsis not subjected to centrifugation prior to contacting said dehydratedadipose tissue of (c).
 28. The method of claim 26, wherein said isolatedpopulation of adipose-derived regenerative cells is prepared in a seconddevice of claim 1 by contacting adipose tissue present in said firstchamber of said second device with collagenase under conditions thatliberate said cells.
 29. The method of claim 28, wherein contacting thedehydrated adipose tissue of (d) with said isolated population ofadipose-derived regenerative cells is performed in the first device.